Closed process for expansion and gene editing of tumor infiltrating lymphocytes and uses of same in immunotherapy

ABSTRACT

The present invention provides improved and/or shortened methods for expanding TILs and producing therapeutic populations of TILs, including novel methods for expanding TIL populations in a closed system that lead to improved efficacy, improved phenotype, and increased metabolic health of the TILs in a shorter time period, while allowing for reduced microbial contamination as well as decreased costs. The methods may comprise gene-editing at least a portion of the TILs to enhance their therapeutic efficacy. Such TILs find use in therapeutic treatment regimens.

FIELD OF THE INVENTION

Methods for expanding tumor infiltrating lymphocytes (TILs) andproducing therapeutic populations of TILs are described herein. Inaddition, methods for gene-editing TILs, and uses of gene-edited TILs inthe treatment of diseases such as cancer are disclosed herein.

BACKGROUND OF THE INVENTION

Treatment of bulky, refractory cancers using adoptive transfer of tumorinfiltrating lymphocytes (TILs) represents a powerful approach totherapy for patients with poor prognoses. Gattinoni, et al., Nat. Rev.Immunol. 2006, 6, 383-393. A large number of TILs are required forsuccessful immunotherapy, and a robust and reliable process is neededfor commercialization. This has been a challenge to achieve because oftechnical, logistical, and regulatory issues with cell expansion.IL-2-based TIL expansion followed by a “rapid expansion process” (REP)has become a preferred method for TIL expansion because of its speed andefficiency. Dudley, et al., Science 2002, 298, 850-54; Dudley, et al.,J. Clin. Oncol. 2005, 23, 2346-57; Dudley, et al., J. Clin. Oncol. 2008,26, 5233-39; Riddell, et al., Science 1992, 257, 238-41; Dudley, et al.,J. Immunother. 2003, 26, 332-42. REP can result in a 1,000-foldexpansion of TILs over a 14-day period, although it requires a largeexcess (e.g., 200-fold) of irradiated allogeneic peripheral bloodmononuclear cells (PBMCs, also known as mononuclear cells (MNCs)), oftenfrom multiple donors, as feeder cells, as well as anti-CD3 antibody(OKT3) and high doses of IL-2. Dudley, et al., J Immunother. 2003, 26,332-42. TILs that have undergone an REP procedure have producedsuccessful adoptive cell therapy following host immunosuppression inpatients with melanoma. Current infusion acceptance parameters rely onreadouts of the composition of TILs (e.g., CD28, CD8, or CD4 positivity)and on fold expansion and viability of the REP product.

Current TIL manufacturing processes are limited by length, cost,sterility concerns, and other factors described herein such that thepotential to commercialize such processes is severely limited, and forthese and other reasons, at the present time no commercial process hasbecome available. There is an urgent need to provide TIL manufacturingprocesses and therapies based on such processes that are appropriate forcommercial scale manufacturing and regulatory approval for use in humanpatients at multiple clinical centers. Moreover, there is a strong needfor more effective TIL therapies that can increase a patient's responserate and response robustness.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods for expanding TILs and producingtherapeutic populations of TILs. According to exemplary embodiments, atleast a portion of the therapeutic population of TILs are gene-edited toenhance their therapeutic effect.

In an embodiment, the present invention provides a method for expandingtumor infiltrating lymphocytes (TILs) into a therapeutic population ofTILs comprising:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a patient by processing a tumor sample obtained from the        patient into multiple tumor fragments;    -   (b) adding the tumor fragments into a closed system;    -   (c) performing a first expansion by culturing the first        population of TILs in a cell culture medium comprising IL-2, and        optionally OKT-3, to produce a second population of TILs,        wherein the first expansion is performed in a closed container        providing a first gas-permeable surface area, wherein the first        expansion is performed for about 3-14 days to obtain the second        population of TILs, wherein the second population of TILs is at        least 50-fold greater in number than the first population of        TILs, and wherein the transition from step (b) to step (c)        occurs without opening the system;    -   (d) performing a second expansion by supplementing the cell        culture medium of the second population of TILs with additional        IL-2, optionally OKT-3, and antigen presenting cells (APCs), to        produce a third population of TILs, wherein the second expansion        is performed for about 7-14 days to obtain the third population        of TILs, wherein the third population of TILs is a therapeutic        population of TILs, wherein the second expansion is performed in        a closed container providing a second gas-permeable surface        area, and wherein the transition from step (c) to step (d)        occurs without opening the system;    -   (e) harvesting the therapeutic population of TILs obtained from        step (d), wherein the transition from step (d) to step (e)        occurs without opening the system;    -   (f) transferring the harvested TIL population from step (e) to        an infusion bag, wherein the transfer from step (e) to (f)        occurs without opening the system; and    -   (g) at any time during the method, gene-editing at least a        portion of the TILs.

In some embodiments, the method further comprises the step ofcryopreserving the infusion bag comprising the harvested TIL populationin step (f) using a cryopreservation process.

In some embodiments, the cryopreservation process is performed using a1:1 ratio of harvested TIL population to cryopreservation media.

In some embodiments, the antigen-presenting cells are peripheral bloodmononuclear cells (PBMCs). In some embodiments, the PBMCs are irradiatedand allogeneic. In some embodiments, the PBMCs are added to the cellculture on any of days 9 through 14 in step (d). In some embodiments,the antigen-presenting cells are artificial antigen-presenting cells.

In some embodiments, the harvesting in step (e) is performed using amembrane-based cell processing system.

In some embodiments, the harvesting in step (e) is performed using aLOVO cell processing system.

In some embodiments, the multiple fragments comprise about 4 to about 50fragments, wherein each fragment has a volume of about 27 mm³.

In some embodiments, the multiple fragments comprise about 30 to about60 fragments with a total volume of about 1300 mm³ to about 1500 mm³.

In some embodiments, the multiple fragments comprise about 50 fragmentswith a total volume of about 1350 mm³.

In some embodiments, the multiple fragments comprise about 50 fragmentswith a total mass of about 1 gram to about 1.5 grams.

In some embodiments, the cell culture medium is provided in a containerselected from the group consisting of a G-container and a Xuri cellbag.

In some embodiments, the cell culture medium in step (d) furthercomprises IL-15 and/or IL-21.

In some embodiments, the IL-2 concentration is about 10,000 IU/mL toabout 5,000 IU/mL.

In some embodiments, the IL-15 concentration is about 500 IU/mL to about100 IU/mL.

In some embodiments, the IL-21 concentration is about 20 IU/mL to about0.5 IU/mL.

In some embodiments, the infusion bag in step (f) is aHypoThermosol-containing infusion bag.

In some embodiments, the cryopreservation media comprisesdimethlysulfoxide (DMSO). In some embodiments, the cryopreservationmedia comprises 7% to 10% dimethlysulfoxide (DMSO).

In some embodiments, the first period in step (c) and the second periodin step (e) are each individually performed within a period of 10 days,11 days, or 12 days.

In some embodiments, the first period in step (c) and the second periodin step (e) are each individually performed within a period of 11 days.

In some embodiments, steps (a) through (f) are performed within a periodof about 10 days to about 22 days.

In some embodiments, steps (a) through (f) are performed within a periodof about 20 days to about 22 days.

In some embodiments, steps (a) through (f) are performed within a periodof about 15 days to about 20 days.

In some embodiments, steps (a) through (f) are performed within a periodof about 10 days to about 20 days.

In some embodiments, steps (a) through (f) are performed within a periodof about 10 days to about 15 days.

In some embodiments, steps (a) through (f) are performed in 22 days orless.

In some embodiments, steps (a) through (f) are performed in 20 days orless.

In some embodiments, steps (a) through (f) are performed in 15 days orless.

In some embodiments, steps (a) through (f) are performed in 10 days orless.

In some embodiments, steps (a) through (f) and cryopreservation areperformed in 22 days or less.

In some embodiments, the therapeutic population of TILs harvested instep (e) comprises sufficient TILs for a therapeutically effectivedosage of the TILs.

In some embodiments, the number of TILs sufficient for a therapeuticallyeffective dosage is from about 2.3×1010 to about 13.7×1010.

In some embodiments, steps (b) through (e) are performed in a singlecontainer, wherein performing steps (b) through (e) in a singlecontainer results in an increase in TIL yield per resected tumor ascompared to performing steps (b) through (e) in more than one container.

In some embodiments, the antigen-presenting cells are added to the TILsduring the second period in step (d) without opening the system.

In some embodiments, the third population of TILs in step (d) providesfor increased efficacy, increased interferon-gamma production, increasedpolyclonality, increased average IP-10, and/or increased average MCP-1when administered to a subject.

In some embodiments, the third population of TILs in step (d) providesfor at least a five-fold or more interferon-gamma production whenadministered to a subject.

In some embodiments, the third population of TILs in step (d) is atherapeutic population of TILs which comprises an increasedsubpopulation of effector T cells and/or central memory T cells relativeto the second population of TILs, wherein the effector T cells and/orcentral memory T cells in the therapeutic population of TILs exhibit oneor more characteristics selected from the group consisting of expressingCD27+, expressing CD28+, longer telomeres, increased CD57 expression,and decreased CD56 expression relative to effector T cells, and/orcentral memory T cells obtained from the second population of cells.

In some embodiments, the effector T cells and/or central memory T cellsobtained from the third population of TILs exhibit increased CD57expression and decreased CD56 expression relative to effector T cellsand/or central memory T cells obtained from the second population ofcells.

In some embodiments, the risk of microbial contamination is reduced ascompared to an open system.

In some embodiments, the TILs from step (g) are infused into a patient.

In some embodiments, the multiple fragments comprise about 4 fragments.

In some embodiments, the cell culture medium further comprises a 4-1BBagonist and/or an OX40 agonist during the first expansion, the secondexpansion, or both.

In some embodiments, the gene-editing is carried out after the 4-1BBagonist and/or the OX40 agonist is introduced into the cell culturemedium.

In some embodiments, the gene-editing is carried out before the 4-1BBagonist and/or the OX40 agonist is introduced into the cell culturemedium.

In some embodiments, the gene-editing is carried out on TILs from one ormore of the first population, the second population, and the thirdpopulation.

In some embodiments, the gene-editing is carried out on TILs from thefirst expansion, or TILs from the second expansion, or both.

In some embodiments, the gene-editing is carried out after the firstexpansion and before the second expansion.

In some embodiments, the gene-editing is carried out before step (c),before step (d), or before step (e).

In some embodiments, the cell culture medium comprises OKT-3 during thefirst expansion and/or during the second expansion, and the gene-editingis carried out before the OKT-3 is introduced into the cell culturemedium.

In some embodiments, the cell culture medium comprises OKT-3 during thefirst expansion and/or during the second expansion, and the gene-editingis carried out after the OKT-3 is introduced into the cell culturemedium.

In some embodiments, the cell culture medium comprises OKT-3 beginningon the start day of the first expansion, and the gene-editing is carriedout after the TILs have been exposed to the OKT-3.

In some embodiments, the gene-editing causes expression of one or moreimmune checkpoint genes to be silenced or reduced in at least a portionof the therapeutic population of TILs.

In some embodiments, the one or more immune checkpoint genes is/areselected from the group comprising PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3),Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA,CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B,TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3,SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST,EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2,and GUCY1B3.

In some embodiments, the one or more immune checkpoint genes is/areselected from the group comprising PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3),Cish, TGFβ, and PKA.

In some embodiments, the gene-editing causes expression of one or moreimmune checkpoint genes to be enhanced in at least a portion of thetherapeutic population of TILs, the immune checkpoint gene(s) beingselected from the group comprising CCR2, CCR4, CCR5, CXCR2, CXCR3,CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-21, the NOTCH 1/2intracellular domain (ICD), and/or the NOTCH ligand mDLL1.

In some embodiments, the gene-editing comprises the use of aprogrammable nuclease that mediates the generation of a double-strand orsingle-strand break at said one or more immune checkpoint genes.

In some embodiments, the gene-editing comprises one or more methodsselected from a CRISPR method, a TALE method, a zinc finger method, anda combination thereof.

In some embodiments, the gene-editing comprises a CRISPR method.

In some embodiments, the CRISPR method is a CRISPR/Cas9 method.

In some embodiments, the gene-editing comprises a TALE method.

In some embodiments, the gene-editing comprises a zinc finger method.

In another embodiment, the present invention provides a method fortreating a subject with cancer, the method comprising administeringexpanded tumor infiltrating lymphocytes (TILs) comprising:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a subject by processing a tumor sample obtained from the        patient into multiple tumor fragments;    -   (b) adding the tumor fragments into a closed system;    -   (c) performing a first expansion by culturing the first        population of TILs in a cell culture medium comprising IL-2, and        optionally OKT-3, to produce a second population of TILs,        wherein the first expansion is performed in a closed container        providing a first gas-permeable surface area, wherein the first        expansion is performed for about 3-14 days to obtain the second        population of TILs, wherein the second population of TILs is at        least 50-fold greater in number than the first population of        TILs, and wherein the transition from step (b) to step (c)        occurs without opening the system;    -   (d) performing a second expansion by supplementing the cell        culture medium of the second population of TILs with additional        IL-2, optionally OKT-3, and antigen presenting cells (APCs), to        produce a third population of TILs, wherein the second expansion        is performed for about 7-14 days to obtain the third population        of TILs, wherein the third population of TILs is a therapeutic        population of TILs, wherein the second expansion is performed in        a closed container providing a second gas-permeable surface        area, and wherein the transition from step (c) to step (d)        occurs without opening the system;    -   (e) harvesting the therapeutic population of TILs obtained from        step (d), wherein the transition from step (d) to step (e)        occurs without opening the system; and    -   (f) transferring the harvested TIL population from step (e) to        an infusion bag, wherein the transfer from step (e) to (f)        occurs without opening the system;    -   (g) optionally cryopreserving the infusion bag comprising the        harvested TIL population from step (f) using a cryopreservation        process;    -   (h) administering a therapeutically effective dosage of the        third population of TILs from the infusion bag in step (g) to        the patient; and    -   (i) at any time during the method steps (a)-(f), gene-editing at        least a portion of the TILs.

In some embodiments, the therapeutic population of TILs harvested instep (e) comprises sufficient TILs for administering a therapeuticallyeffective dosage of the TILs in step (h).

In some embodiments, the number of TILs sufficient for administering atherapeutically effective dosage in step (h) is from about 2.3×1010 toabout 13.7×1010.

In some embodiments, the antigen presenting cells (APCs) are PBMCs.

In some embodiments, the PBMCs are added to the cell culture on any ofdays 9 through 14 in step (d).

In some embodiments, prior to administering a therapeutically effectivedosage of TIL cells in step (h), a non-myeloablative lymphodepletionregimen has been administered to the patient.

In some embodiments, the non-myeloablative lymphodepletion regimencomprises the steps of administration of cyclophosphamide at a dose of60 mg/m²/day for two days followed by administration of fludarabine at adose of 25 mg/m²/day for five days.

In some embodiments, the method further comprises the step of treatingthe patient with a high-dose IL-2 regimen starting on the day afteradministration of the TIL cells to the patient in step (h).

In some embodiments, the high-dose IL-2 regimen comprises 600,000 or720,000 IU/kg administered as a 15-minute bolus intravenous infusionevery eight hours until tolerance.

In some embodiments, the third population of TILs in step (d) is atherapeutic population of TILs which comprises an increasedsubpopulation of effector T cells and/or central memory T cells relativeto the second population of TILs, wherein the effector T cells and/orcentral memory T cells in the therapeutic population of TILs exhibit oneor more characteristics selected from the group consisting of expressingCD27+, expressing CD28+, longer telomeres, increased CD57 expression,and decreased CD56 expression relative to effector T cells, and/orcentral memory T cells obtained from the second population of cells.

In some embodiments, the effector T cells and/or central memory T cellsin the therapeutic population of TILs exhibit increased CD57 expressionand decreased CD56 expression relative to effector T cells and/orcentral memory T cells obtained from the second population of cells.

In some embodiments, the cancer is selected from the group consisting ofmelanoma, ovarian cancer, cervical cancer, non-small-cell lung cancer(NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused byhuman papilloma virus, head and neck cancer (including head and necksquamous cell carcinoma (HNSCC)), renal cancer, and renal cellcarcinoma.

In some embodiments, the cancer is selected from the group consisting ofmelanoma, HNSCC, cervical cancers, and NSCLC.

In some embodiments, the cancer is melanoma.

In some embodiments, the cancer is HNSCC.

In some embodiments, the cancer is a cervical cancer.

In some embodiments, the cancer is NSCLC.

In some embodiments, the cell culture medium further comprises a 4-1BBagonist and/or an OX40 agonist during the first expansion, the secondexpansion, or both.

In some embodiments, the gene-editing is carried out after the 4-1BBagonist and/or the OX40 agonist is introduced into the cell culturemedium.

In some embodiments, the gene-editing is carried out before the 4-1BBagonist and/or the OX40 agonist is introduced into the cell culturemedium.

In some embodiments, the gene-editing is carried out on TILs from one ormore of the first population, the second population, and the thirdpopulation.

In some embodiments, the gene-editing is carried out on TILs from thefirst expansion, or TILs from the second expansion, or both.

In some embodiments, the gene-editing is carried out after the firstexpansion and before the second expansion.

In some embodiments, the gene-editing is carried out before step (c),before step (d), or before step (e).

In some embodiments, the cell culture medium comprises OKT-3 during thefirst expansion and/or during the second expansion, and the gene-editingis carried out before the OKT-3 is introduced into the cell culturemedium.

In some embodiments, the cell culture medium comprises OKT-3 during thefirst expansion and/or during the second expansion, and the gene-editingis carried out after the OKT-3 is introduced into the cell culturemedium.

In some embodiments, the cell culture medium comprises OKT-3 beginningon the start day of the first expansion, and the gene-editing is carriedout after the TILs have been exposed to the OKT-3.

In some embodiments, the gene-editing causes expression of one or moreimmune checkpoint genes to be silenced or reduced in at least a portionof the therapeutic population of TILs.

In some embodiments, the one or more immune checkpoint genes is/areselected from the group comprising PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3),Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA,CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B,TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3,SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST,EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2,and GUCY1B3.

In some embodiments, the one or more immune checkpoint genes is/areselected from the group comprising PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3),Cish, TGFβ, and PKA.

In some embodiments, the gene-editing causes expression of one or moreimmune checkpoint genes to be enhanced in at least a portion of thetherapeutic population of TILs, the immune checkpoint gene(s) beingselected from the group comprising CCR2, CCR4, CCR5, CXCR2, CXCR3,CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-21, the NOTCH 1/2intracellular domain (ICD), and/or the NOTCH ligand mDLL1.

In some embodiments, the gene-editing comprises the use of aprogrammable nuclease that mediates the generation of a double-strand orsingle-strand break at said one or more immune checkpoint genes.

In some embodiments, the gene-editing comprises one or more methodsselected from a CRISPR method, a TALE method, a zinc finger method, anda combination thereof.

In some embodiments, the gene-editing comprises a CRISPR method.

In some embodiments, the CRISPR method is a CRISPR/Cas9 method.

In some embodiments, the gene-editing comprises a TALE method.

In some embodiments, the gene-editing comprises a zinc finger method.

In another embodiment, the present invention provides a method forexpanding tumor infiltrating lymphocytes (TILs) into a therapeuticpopulation of TILs comprising:

-   -   (a) adding processed tumor fragments from a tumor resected from        a patient into a closed system to obtain a first population of        TILs;    -   (b) performing a first expansion by culturing the first        population of TILs in a cell culture medium comprising IL-2, and        optionally OKT-3, to produce a second population of TILs,        wherein the first expansion is performed in a closed container        providing a first gas-permeable surface area, wherein the first        expansion is performed for about 3-14 days to obtain the second        population of TILs, wherein the second population of TILs is at        least 50-fold greater in number than the first population of        TILs, and wherein the transition from step (a) to step (b)        occurs without opening the system;    -   (c) performing a second expansion by supplementing the cell        culture medium of the second population of TILs with additional        IL-2, optionally OKT-3, and antigen presenting cells (APCs), to        produce a third population of TILs, wherein the second expansion        is performed for about 7-14 days to obtain the third population        of TILs, wherein the third population of TILs is a therapeutic        population of TILs, wherein the second expansion is performed in        a closed container providing a second gas-permeable surface        area, and wherein the transition from step (b) to step (c)        occurs without opening the system;    -   (d) harvesting the therapeutic population of TILs obtained from        step (c), wherein the transition from step (c) to step (d)        occurs without opening the system;    -   (e) transferring the harvested TIL population from step (d) to        an infusion bag, wherein the transfer from step (d) to (e)        occurs without opening the system; and    -   (f) at any time during the method, gene-editing at least a        portion of the TILs.

In some embodiments, the therapeutic population of TILs harvested instep (d) comprises sufficient TILs for a therapeutically effectivedosage of the TILs.

In some embodiments, the number of TILs sufficient for a therapeuticallyeffective dosage is from about 2.3×1010 to about 13.7×1010.

In some embodiments, the method further comprises the step ofcryopreserving the infusion bag comprising the harvested TIL populationusing a cryopreservation process.

In some embodiments, the cryopreservation process is performed using a1:1 ratio of harvested TIL population to cryopreservation media.

In some embodiments, the antigen-presenting cells are peripheral bloodmononuclear cells (PBMCs).

In some embodiments, the PBMCs are irradiated and allogeneic.

The method according to claim 68, wherein the PBMCs are added to thecell culture on any of days 9 through 14 in step (c).

In some embodiments, the antigen-presenting cells are artificialantigen-presenting cells.

In some embodiments, the harvesting in step (d) is performed using aLOVO cell processing system.

In some embodiments, the multiple fragments comprise about 4 to about 50fragments, wherein each fragment has a volume of about 27 mm³.

In some embodiments, the multiple fragments comprise about 30 to about60 fragments with a total volume of about 1300 mm³ to about 1500 mm³.

In some embodiments, the multiple fragments comprise about 50 fragmentswith a total volume of about 1350 mm³.

In some embodiments, the multiple fragments comprise about 50 fragmentswith a total mass of about 1 gram to about 1.5 grams.

In some embodiments, the multiple fragments comprise about 4 fragments.

In some embodiments, the second cell culture medium is provided in acontainer selected from the group consisting of a G-container and a Xuricellbag.

In some embodiments, the infusion bag in step (e) is aHypoThermosol-containing infusion bag.

In some embodiments, the first period in step (b) and the second periodin step (c) are each individually performed within a period of 10 days,11 days, or 12 days.

In some embodiments, the first period in step (b) and the second periodin step (c) are each individually performed within a period of 11 days.

In some embodiments, steps (a) through (e) are performed within a periodof about 10 days to about 22 days.

In some embodiments, steps (a) through (e) are performed within a periodof about 10 days to about 20 days.

In some embodiments, steps (a) through (e) are performed within a periodof about 10 days to about 15 days.

In some embodiments, steps (a) through (e) are performed in 22 days orless.

In some embodiments, steps (a) through (e) and cryopreservation areperformed in 22 days or less.

In some embodiments, steps (b) through (e) are performed in a singlecontainer, wherein performing steps (b) through (e) in a singlecontainer results in an increase in TIL yield per resected tumor ascompared to performing steps (b) through (e) in more than one container.

In some embodiments, the antigen-presenting cells are added to the TILsduring the second period in step (c) without opening the system.

In some embodiments, the third population of TILs in step (d) is atherapeutic population of TILs which comprises an increasedsubpopulation of effector T cells and/or central memory T cells relativeto the second population of TILs, wherein the effector T cells and/orcentral memory T cells obtained in the therapeutic population of TILsexhibit one or more characteristics selected from the group consistingof expressing CD27+, expressing CD28+, longer telomeres, increased CD57expression, and decreased CD56 expression relative to effector T cells,and/or central memory T cells obtained from the second population ofcells.

In some embodiments, the effector T cells and/or central memory T cellsobtained in the therapeutic population of TILs exhibit increased CD57expression and decreased CD56 expression relative to effector T cells,and/or central memory T cells obtained from the second population ofcells.

In some embodiments, the risk of microbial contamination is reduced ascompared to an open system.

In some embodiments, the TILs from step (e) are infused into a patient.

In some embodiments, the closed container comprises a single bioreactor.

In some embodiments, the closed container comprises a G-REX-10.

In some embodiments, the closed container comprises a G-REX-100.

In some embodiments, at step (d) the antigen presenting cells (APCs) areadded to the cell culture of the second population of TILs at a APC:TILratio of 25:1 to 100:1.

In some embodiments, the cell culture has a ratio of 2.5×10⁹ APCs to100×10⁶ TILs.

In some embodiments, at step (c) the antigen presenting cells (APCs) areadded to the cell culture of the second population of TILs at a APC:TILratio of 25:1 to 100:1.

In some embodiments, the cell culture has ratio of 2.5×10⁹ APCs to100×10⁶ TILs.

In some embodiments, the cell culture medium further comprises a 4-1BBagonist and/or an OX40 agonist during the first expansion, the secondexpansion, or both.

In some embodiments, the gene-editing is carried out after the 4-1BBagonist and/or the OX40 agonist is introduced into the cell culturemedium.

In some embodiments, the gene-editing is carried out before the 4-1BBagonist and/or the OX40 agonist is introduced into the cell culturemedium.

In some embodiments, the gene-editing is carried out on TILs from one ormore of the first population, the second population, and the thirdpopulation.

In some embodiments, the gene-editing is carried out on TILs from thefirst expansion, or TILs from the second expansion, or both.

In some embodiments, the gene-editing is carried out after the firstexpansion and before the second expansion.

In some embodiments, the gene-editing is carried out before step (b),before step (c), or before step (d).

In some embodiments, the cell culture medium comprises OKT-3 during thefirst expansion and/or during the second expansion, and the gene-editingis carried out before the OKT-3 is introduced into the cell culturemedium.

In some embodiments, the cell culture medium comprises OKT-3 during thefirst expansion and/or during the second expansion, and the gene-editingis carried out after the OKT-3 is introduced into the cell culturemedium.

In some embodiments, the cell culture medium comprises OKT-3 beginningon the start day of the first expansion, and the gene-editing is carriedout after the TILs have been exposed to the OKT-3.

In some embodiments, the gene-editing causes expression of one or moreimmune checkpoint genes to be silenced or reduced in at least a portionof the therapeutic population of TILs.

In some embodiments, the one or more immune checkpoint genes is/areselected from the group comprising PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3),Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA,CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B,TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3,SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST,EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2,and GUCY1B3.

In some embodiments, the one or more immune checkpoint genes is/areselected from the group comprising PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3),Cish, TGFβ, and PKA.

In some embodiments, the gene-editing causes expression of one or moreimmune checkpoint genes to be enhanced in at least a portion of thetherapeutic population of TILs, the immune checkpoint gene(s) beingselected from the group comprising CCR2, CCR4, CCR5, CXCR2, CXCR3,CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-21, the NOTCH 1/2intracellular domain (ICD), and/or the NOTCH ligand mDLL1.

In some embodiments, the gene-editing comprises the use of aprogrammable nuclease that mediates the generation of a double-strand orsingle-strand break at said one or more immune checkpoint genes.

In some embodiments, the gene-editing comprises one or more methodsselected from a CRISPR method, a TALE method, a zinc finger method, anda combination thereof.

In some embodiments, the gene-editing comprises a CRISPR method.

In some embodiments, the CRISPR method is a CRISPR/Cas9 method.

In some embodiments, the gene-editing comprises a TALE method.

In some embodiments, the gene-editing comprises a zinc finger method.

In another embodiment, the present invention provides a population oftherapeutic TILs that have been expanded in accordance with any of theexpansion methods described herein (e.g., for use in the treatment of asubject's cancer), wherein the population of therapeutic TILs has beenpermanently gene-edited.

In another embodiment, the present invention provides a population ofexpanded TILs for use in the treatment of a subject with cancer, whereinthe population of expanded TILs is a third population of TILs obtainableby a method comprising:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a subject by processing a tumor sample obtained from the        patient into multiple tumor fragments;    -   (b) adding the tumor fragments into a closed system;    -   (c) performing a first expansion by culturing the first        population of TILs in a cell culture medium comprising IL-2, and        optionally OKT-3, to produce a second population of TILs,        wherein the first expansion is performed in a closed container        providing a first gas-permeable surface area, wherein the first        expansion is performed for about 3-14 days to obtain the second        population of TILs, wherein the second population of TILs is at        least 50-fold greater in number than the first population of        TILs, and wherein the transition from step (b) to step (c)        occurs without opening the system;    -   (d) performing a second expansion by supplementing the cell        culture medium of the second population of TILs with additional        IL-2, optionally OKT-3, and antigen presenting cells (APCs), to        produce a third population of TILs, wherein the second expansion        is performed for about 7-14 days to obtain the third population        of TILs, wherein the third population of TILs is a therapeutic        population of TILs, wherein the second expansion is performed in        a closed container providing a second gas-permeable surface        area, and wherein the transition from step (c) to step (d)        occurs without opening the system;    -   (e) harvesting the therapeutic population of TILs obtained from        step (d), wherein the transition from step (d) to step (e)        occurs without opening the system;    -   (f) transferring the harvested TIL population from step (e) to        an infusion bag, wherein the transfer from step (e) to (f)        occurs without opening the system;    -   (g) optionally cryopreserving the infusion bag comprising the        harvested TIL population from step (f) using a cryopreservation        process; and    -   (h) at any time during the method, gene-editing at least a        portion of the TILs.

In some embodiments, the above method further comprises one or morefeatures recited in any of the methods and compositions describedherein.

In some embodiments, the cell culture medium further comprises a 4-1BBagonist and/or an OX40 agonist during the first expansion, the secondexpansion, or both.

In some embodiments, the gene-editing is carried out after the 4-1BBagonist and/or the OX40 agonist is introduced into the cell culturemedium.

In some embodiments, the gene-editing is carried out before the 4-1BBagonist and/or the OX40 agonist is introduced into the cell culturemedium.

In some embodiments, the gene-editing is carried out on TILs from one ormore of the first population, the second population, and the thirdpopulation.

In some embodiments, the gene-editing is carried out on TILs from thefirst expansion, or TILs from the second expansion, or both.

In some embodiments, the gene-editing is carried out after the firstexpansion and before the second expansion.

In some embodiments, the gene-editing is carried out before step (c),before step (d), or before step (e).

In some embodiments, the cell culture medium comprises OKT-3 during thefirst expansion and/or during the second expansion, and the gene-editingis carried out before the OKT-3 is introduced into the cell culturemedium.

In some embodiments, the cell culture medium comprises OKT-3 during thefirst expansion and/or during the second expansion, and the gene-editingis carried out after the OKT-3 is introduced into the cell culturemedium.

In some embodiments, the cell culture medium comprises OKT-3 beginningon the start day of the first expansion, and the gene-editing is carriedout after the TILs have been exposed to the OKT-3.

In some embodiments, the gene-editing causes expression of one or moreimmune checkpoint genes to be silenced or reduced in at least a portionof the therapeutic population of TILs.

In some embodiments, the one or more immune checkpoint genes is/areselected from the group comprising PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3),Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA,CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B,TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3,SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST,EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2,and GUCY1B3.

In some embodiments, the one or more immune checkpoint genes is/areselected from the group comprising PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3),Cish, TGFβ, and PKA.

In some embodiments, the gene-editing causes expression of one or moreimmune checkpoint genes to be enhanced in at least a portion of thetherapeutic population of TILs, the immune checkpoint gene(s) beingselected from the group comprising CCR2, CCR4, CCR5, CXCR2, CXCR3,CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-21, the NOTCH 1/2intracellular domain (ICD), and/or the NOTCH ligand mDLL1.

In some embodiments, the gene-editing comprises the use of aprogrammable nuclease that mediates the generation of a double-strand orsingle-strand break at said one or more immune checkpoint genes.

In some embodiments, the gene-editing comprises one or more methodsselected from a CRISPR method, a TALE method, a zinc finger method, anda combination thereof.

In some embodiments, the gene-editing comprises a CRISPR method.

In some embodiments, the CRISPR method is a CRISPR/Cas9 method.

In some embodiments, the gene-editing comprises a TALE method.

In some embodiments, the gene-editing comprises a zinc finger method.

In another embodiment, the present invention provides a method forexpanding tumor infiltrating lymphocytes (TILs) into a therapeuticpopulation of TILs comprising:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a patient by processing a tumor sample obtained from the        patient into multiple tumor fragments;    -   (b) adding the tumor fragments into a closed system;    -   (c) performing a first expansion by culturing the first        population of TILs in a cell culture medium comprising IL-2 and        optionally comprising OKT-3 and/or a 4-1BB agonist antibody for        about 2 to 5 days;    -   (d) optionally adding OKT-3, to produce a second population of        TILs, wherein the first expansion is performed in a closed        container providing a first gas-permeable surface area, wherein        the first expansion is performed for about 1 to 3 days to obtain        the second population of TILs, wherein the second population of        TILs is at least 50-fold greater in number than the first        population of TILs, and wherein the transition from step (c) to        step (d) occurs without opening the system;    -   (e) performing a sterile electroporation step on the second        population of TILs, wherein the sterile electroporation step        mediates the transfer of at least one gene editor;    -   (f) resting the second population of TILs for about 1 day;    -   (g) performing a second expansion by supplementing the cell        culture medium of the second population of TILs with additional        IL-2, optionally OKT-3 antibody, optionally an OX40 antibody,        and antigen presenting cells (APCs), to produce a third        population of TILs, wherein the second expansion is performed        for about 7 to 11 days to obtain the third population of TILs,        wherein the second expansion is performed in a closed container        providing a second gas-permeable surface area, and wherein the        transition from step (f) to step (g) occurs without opening the        system;    -   (h) harvesting the therapeutic population of TILs obtained from        step (g) to provide a harvested TIL population, wherein the        transition from step (g) to step (h) occurs without opening the        system, wherein the harvested population of TILs is a        therapeutic population of TILs;    -   (i) transferring the harvested TIL population to an infusion        bag, wherein the transfer from step (h) to (i) occurs without        opening the system; and    -   (j) cryopreserving the harvested TIL population using a        dimethylsulfoxide-based cryopreservation medium, wherein the        electroporation step comprises the delivery of a Clustered        Regularly Interspersed Short Palindromic Repeat (CRISPR) system,        a Transcription Activator-Like Effector (TALE) system, or a zinc        finger system for inhibiting the expression of a molecule        selected from the group consisting of PD-1, LAG-3, TIM-3,        CTLA-4, TIGIT, CISH, TGFβR2, PRA, CBLB, BAFF (BR3), and        combinations thereof.

In another embodiment, the present invention provides a method fortreating a subject with cancer, the method comprising administeringexpanded tumor infiltrating lymphocytes (TILs) comprising:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a patient by processing a tumor sample obtained from the        patient into multiple tumor fragments;    -   (b) adding the tumor fragments into a closed system;    -   (c) performing a first expansion by culturing the first        population of TILs in a cell culture medium comprising IL-2 and        optionally comprising OKT-3 and/or a 4-1BB agonist antibody for        about 2 to 5 days;    -   (d) optionally adding OKT-3, to produce a second population of        TILs, wherein the first expansion is performed in a closed        container providing a first gas-permeable surface area, wherein        the first expansion is performed for about 1 to 3 days to obtain        the second population of TILs, wherein the second population of        TILs is at least 50-fold greater in number than the first        population of TILs, and wherein the transition from step (c) to        step (d) occurs without opening the system;    -   (e) performing a sterile electroporation step on the second        population of TILs, wherein the sterile electroporation step        mediates the transfer of at least one gene editor;    -   (f) resting the second population of TILs for about 1 day;    -   (g) performing a second expansion by supplementing the cell        culture medium of the second population of TILs with additional        IL-2, optionally OKT-3 antibody, optionally an OX40 antibody,        and antigen presenting cells (APCs), to produce a third        population of TILs, wherein the second expansion is performed        for about 7 to 11 days to obtain the third population of TILs,        wherein the second expansion is performed in a closed container        providing a second gas-permeable surface area, and wherein the        transition from step (f) to step (g) occurs without opening the        system;    -   (h) harvesting the therapeutic population of TILs obtained from        step (g) to provide a harvested TIL population, wherein the        transition from step (g) to step (h) occurs without opening the        system, wherein the harvested population of TILs is a        therapeutic population of TILs;    -   (i) transferring the harvested TIL population to an infusion        bag, wherein the transfer from step (h) to (i) occurs without        opening the system;    -   (j) cryopreserving the harvested TIL population using a        dimethylsulfoxide-based cryopreservation medium; and    -   (k) administering a therapeutically effective dosage of the        harvested TIL population from the infusion bag to the patient;

wherein the electroporation step comprises the delivery of a ClusteredRegularly Interspersed Short Palindromic Repeat (CRISPR) system, aTranscription Activator-Like Effector (TALE) system, or a zinc fingersystem for inhibiting the expression of a molecule selected from thegroup consisting of PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGFβR2,PRA, CBLB, BAFF (BR3), and combinations thereof.

In another embodiment, the present invention provides a population ofexpanded TILs for use in the treatment of a subject with cancer, whereinthe population of expanded TILs is a harvested population of TILsobtainable by a method comprising:

-   -   (a) obtaining a first population of TILs from a tumor resected        from a patient by processing a tumor sample obtained from the        patient into multiple tumor fragments;    -   (b) adding the tumor fragments into a closed system;    -   (c) performing a first expansion by culturing the first        population of TILs in a cell culture medium comprising IL-2 and        optionally comprising OKT-3 and/or a 4-1BB agonist antibody for        about 2 to 5 days;    -   (d) optionally adding OKT-3, to produce a second population of        TILs, wherein the first expansion is performed in a closed        container providing a first gas-permeable surface area, wherein        the first expansion is performed for about 1 to 3 days to obtain        the second population of TILs, wherein the second population of        TILs is at least 50-fold greater in number than the first        population of TILs, and wherein the transition from step (c) to        step (d) occurs without opening the system;    -   (e) performing a sterile electroporation step on the second        population of TILs, wherein the sterile electroporation step        mediates the transfer of at least one gene editor;    -   (f) resting the second population of TILs for about 1 day;    -   (g) performing a second expansion by supplementing the cell        culture medium of the second population of TILs with additional        IL-2, optionally OKT-3 antibody, optionally an OX40 antibody,        and antigen presenting cells (APCs), to produce a third        population of TILs, wherein the second expansion is performed        for about 7 to 11 days to obtain the third population of TILs,        wherein the second expansion is performed in a closed container        providing a second gas-permeable surface area, and wherein the        transition from step (f) to step (g) occurs without opening the        system;    -   (h) harvesting the therapeutic population of TILs obtained from        step (g) to provide a harvested TIL population, wherein the        transition from step (g) to step (h) occurs without opening the        system, wherein the harvested population of TILs is a        therapeutic population of TILs;    -   (i) transferring the harvested TIL population to an infusion        bag, wherein the transfer from step (h) to (i) occurs without        opening the system; and    -   (j) cryopreserving the harvested TIL population using a        dimethylsulfoxide-based cryopreservation medium,        wherein the electroporation step comprises the delivery of a        Clustered Regularly Interspersed Short Palindromic Repeat        (CRISPR) system, a Transcription Activator-Like Effector (TALE)        system, or a zinc finger system for inhibiting the expression of        a molecule selected from the group consisting of PD-1, LAG-3,        TIM-3, CTLA-4, TIGIT, CISH, TGFβR2, PRA, CBLB, BAFF (BR3), and        combinations thereof.

In some embodiments, the method comprises performing the first expansionby culturing the first population of TILs in a cell culture mediumcomprising IL-2, OKT-3 and a 4-1BB agonist antibody, wherein the OKT-3and the 4-1BB agonist antibody are optionally present in the cellculture medium beginning on Day 0 or Day 1.

In another embodiment, the present invention provides a cryopreservationcomposition comprising the population of TILs for use to treat a subjectwith cancer, a cryoprotectant medium comprising DMSO, and an electrolytesolution.

In some embodiments, the cryopreservation composition may furthercomprise one or more stabilizers (e.g., HSA) and one or more lymphocytegrowth factors (e.g., IL-2).

In some embodiments, the cryoprotectant medium comprising DMSO and theelectrolyte solution are present in a ratio of about 1.1:1 to about1:1.1.

In some embodiments, the cryopreservation composition comprises thecryoprotectant medium comprising DMSO in an amount of about 30 mL toabout 70 mL, the electrolyte solution in an amount of about 30 mL toabout 70 mL, HSA in an amount of about 0.1 g to about 1.0 g, and IL-2 inan amount of about 0.001 mg to about 0.005 mg.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Shows a diagram of an embodiment of process 2A, a 22-day processfor TIL manufacturing.

FIG. 2: Shows a comparison between the 1C process and an embodiment ofthe 2A process for TIL manufacturing.

FIG. 3: Shows the 1C process timeline.

FIG. 4: Shows the process of an embodiment of TIL therapy using process2A for TIL manufacturing, including administration and co-therapy steps,for higher cell counts.

FIG. 5: Shows the process of an embodiment of TIL therapy using process2A for TIL manufacturing, including administration and co-therapy steps,for lower cell counts.

FIG. 6: Shows a detailed schematic for an embodiment of the 2A process.

FIGS. 7a, 7b and 7c : Depict the major steps of an embodiment of process2A including the cryopreservation steps.

FIG. 8: Depicts the clinical trial design including cohorts treated withprocess 1C and an embodiment of process 2A.

FIG. 9: Exemplary Process 2A chart providing an overview of Steps Athrough F.

FIG. 10: Process Flow Chart on Process 2A Data Collection Plan

FIG. 11: Scheme of on exemplary embodiment of the Rapid ExpansionProtocol (REP). Upon arrival the tumor is fragmented, placed into G-Rexflasks with IL-2 for TIL expansion (pre-REP expansion), for 11 days. Forthe triple cocktail studies, IL-2/IL-15/IL-21 is added at the initiationof the pre-REP. For the Rapid Expansion Protocol (REP), TIL are culturedwith feeders and OKT3 for REP expansion for an additional 11 days.

FIG. 12: Shows a diagram of an embodiment of process 2A, a 22-dayprocess for TIL manufacturing.

FIG. 13: Comparison table of Steps A through F from exemplaryembodiments of process 1C and process 2A.

FIG. 14: Detailed comparison of an embodiment of process 1C and anembodiment of process 2A.

FIG. 15: Detailed scheme of an embodiment of a TIL therapy process.

FIG. 16: Depiction of an embodiment of a cryopreserved TIL manufacturingprocess (22 days).

FIG. 17: Table of process improvements from Gen 1 to Gen 2.

FIG. 18: An embodiment of a TIL manufacturing process of the presentinvention.

FIG. 19: Process Flow Chart of Process 2A.

FIG. 20: Depiction of an embodiment of a TIL manufacturing processincluding electroporation step for use with gene-editing processes(including TALEN, zinc finger nuclease, and CRISPR methods as describedherein).

FIG. 21: Depiction of embodiments of TIL manufacturing processesincluding electroporation step for use with gene-editing processes(including TALEN, zinc finger nuclease, and CRISPR methods as describedherein).

FIG. 22: Depiction of the structures I-A and I-B, the cylinders refer toindividual polypeptide binding domains. Structures I-A and I-B comprisethree linearly-linked TNFRSF binding domains derived from e.g., 4-1BBLor an antibody that binds 4-1BB, which fold to form a trivalent protein,which is then linked to a second trivalent protein through IgG1-Fc(including CH3 and CH2 domains) is then used to link two of thetrivalent proteins together through disulfide bonds (small elongatedovals), stabilizing the structure and providing an agonists capable ofbringing together the intracellular signaling domains of the sixreceptors and signaling proteins to form a signaling complex. The TNFRSFbinding domains denoted as cylinders may be scFv domains comprising,e.g., a VH and a VL chain connected by a linker that may comprisehydrophilic residues and Gly and Ser sequences for flexibility, as wellas Glu and Lys for solubility.

FIG. 23: Depiction of a TALEN construct that targets exon 2 of the Pdcd1gene.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

Adoptive cell therapy utilizing TILs cultured ex vivo by the RapidExpansion Protocol (REP) has produced successful adoptive cell therapyfollowing host immunosuppression in patients with melanoma. Currentinfusion acceptance parameters rely on readouts of the composition ofTILs (e.g., CD28, CD8, or CD4 positivity) and on the numerical folds ofexpansion and viability of the REP product.

Current REP protocols give little insight into the health of the TILthat will be infused into the patient. T cells undergo a profoundmetabolic shift during the course of their maturation from naïve toeffector T cells (see Chang, et al., Nat. Immunol. 2016, 17, 364, herebyexpressly incorporated in its entirety, and in particular for thediscussion and markers of anaerobic and aerobic metabolism). Forexample, naïve T cells rely on mitochondrial respiration to produce ATP,while mature, healthy effector T cells such as TIL are highlyglycolytic, relying on aerobic glycolysis to provide the bioenergeticssubstrates they require for proliferation, migration, activation, andanti-tumor efficacy.

Previous papers report that limiting glycolysis and promotingmitochondrial metabolism in TILs prior to transfer is desirable as cellsthat are relying heavily on glycolysis will suffer nutrient deprivationupon adoptive transfer which results in a majority of the transferredcells dying. Thus, the art teaches that promoting mitochondrialmetabolism might promote in vivo longevity and in fact suggests usinginhibitors of glycolysis before induction of the immune response. SeeChang, et al., Nat. Immunol. 2016, 17(364).

The present invention is further directed in some embodiments to methodsfor evaluating and quantifying this increase in metabolic health. Thus,the present invention provides methods of assaying the relative healthof a TIL population using one or more general evaluations of metabolism,including, but not limited to, rates and amounts of glycolysis,oxidative phosphorylation, spare respiratory capacity (SRC), andglycolytic reserve.

Furthermore, the present invention is further directed in someembodiments to methods for evaluating and quantifying this increase inmetabolic health. Thus, the present invention provides methods ofassaying the relative health of a TIL population using one or moregeneral evaluations of metabolism, including, but not limited to, ratesand amounts of glycolysis, oxidative phosphorylation, spare respiratorycapacity (SRC), and glycolytic reserve.

In addition, optional additional evaluations include, but are notlimited to, ATP production, mitochondrial mass and glucose uptake.

The present invention is further directed in some embodiments toenhancing the therapeutic effect of TILs with the use of gene editingtechnology. While adoptive transfer of tumor infiltrating lymphocytes(TILs) offers a promising and effective therapy, there is a strong needfor more effective TIL therapies that can increase a patient's responserate and response robustness. As described herein, embodiments of thepresent invention provide methods for expanding TILs into a therapeuticpopulation that is gene-edited to provide an enhanced therapeuticeffect.

II. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. All patents and publicationsreferred to herein are incorporated by reference in their entireties.

The term “in vivo” refers to an event that takes place in a subject'sbody.

The term “in vitro” refers to an event that takes places outside of asubject's body.

In vitro assays encompass cell-based assays in which cells alive or deadare employed and may also encompass a cell-free assay in which no intactcells are employed.

The term “ex vivo” refers to an event which involves treating orperforming a procedure on a cell, tissue and/or organ which has beenremoved from a subject's body.

Aptly, the cell, tissue and/or organ may be returned to the subject'sbody in a method of surgery or treatment.

The term “rapid expansion” means an increase in the number ofantigen-specific TILs of at least about 3-fold (or 4-, 5-, 6-, 7-, 8-,or 9-fold) over a period of a week, more preferably at least about10-fold (or 20-, 30-, 40-, 50-, 60-, 70-, 80-, or 90-fold) over a periodof a week, or most preferably at least about 100-fold over a period of aweek. A number of rapid expansion protocols are outlined below.

By “tumor infiltrating lymphocytes” or “TILs” herein is meant apopulation of cells originally obtained as white blood cells that haveleft the bloodstream of a subject and migrated into a tumor. TILsinclude, but are not limited to, CD8⁺ cytotoxic T cells (lymphocytes),Th1 and Th17 CD4⁺ T cells, natural killer cells, dendritic cells and M1macrophages. TILs include both primary and secondary TILs. “PrimaryTILs” are those that are obtained from patient tissue samples asoutlined herein (sometimes referred to as “freshly harvested”), and“secondary TILs” are any TIL cell populations that have been expanded orproliferated as discussed herein, including, but not limited to bulkTILs and expanded TILs (“REP TILs” or “post-REP TILs”). TIL cellpopulations can include genetically modified TILs.

By “population of cells” (including TILs) herein is meant a number ofcells that share common traits. In general, populations generally rangefrom 1×10⁶ to 1×10¹⁰ in number, with different TIL populationscomprising different numbers. For example, initial growth of primaryTILs in the presence of IL-2 results in a population of bulk TILs ofroughly 1×10¹ cells. REP expansion is generally done to providepopulations of 1.5×10′ to 1.5×10¹⁰ cells for infusion.

By “cryopreserved TILs” herein is meant that TILs, either primary, bulk,or expanded (REP TILs), are treated and stored in the range of about−150° C. to −60° C. General methods for cryopreservation are alsodescribed elsewhere herein, including in the Examples. For clarity,“cryopreserved TILs” are distinguishable from frozen tissue sampleswhich may be used as a source of primary TILs.

By “thawed cryopreserved TILs” herein is meant a population of TILs thatwas previously cryopreserved and then treated to return to roomtemperature or higher, including but not limited to cell culturetemperatures or temperatures wherein TILs may be administered to apatient.

TILs can generally be defined either biochemically, using cell surfacemarkers, or functionally, by their ability to infiltrate tumors andeffect treatment. TILs can be generally categorized by expressing one ormore of the following biomarkers: CD4, CD8, TCR αβ, CD27, CD28, CD56,CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally and alternatively, TILscan be functionally defined by their ability to infiltrate solid tumorsupon reintroduction into a patient.

The term “cryopreservation media” or “cryopreservation medium” refers toany medium that can be used for cryopreservation of cells. Such mediacan include media comprising 7% to 10% DMSO. Exemplary media includeCryoStor CS10, Hyperthermasol, as well as combinations thereof. The term“CS10” refers to a cryopreservation medium which is obtained fromStemcell Technologies or from Biolife Solutions. The CS10 medium may bereferred to by the trade name “CryoStor® CS10”. The CS10 medium is aserum-free, animal component-free medium which comprises DMSO.

The term “central memory T cell” refers to a subset of T cells that inthe human are CD45R0+ and constitutively express CCR7 (CCR7^(hi)) andCD62L (CD62^(hi)). The surface phenotype of central memory T cells alsoincludes TCR, CD3, CD127 (IL-7R), and IL-15R. Transcription factors forcentral memory T cells include BCL-6, BCL-6B, MBD2, and BMI1. Centralmemory T cells primarily secret IL-2 and CD40L as effector moleculesafter TCR triggering. Central memory T cells are predominant in the CD4compartment in blood, and in the human are proportionally enriched inlymph nodes and tonsils.

The term “effector memory T cell” refers to a subset of human ormammalian T cells that, like central memory T cells, are CD45R0+, buthave lost the constitutive expression of CCR7 (CCR7^(lo)) and areheterogeneous or low for CD62L expression (CD62L^(lo)). The surfacephenotype of central memory T cells also includes TCR, CD3, CD127(IL-7R), and IL-15R. Transcription factors for central memory T cellsinclude BLIMP1. Effector memory T cells rapidly secret high levels ofinflammatory cytokines following antigenic stimulation, includinginterferon-γ, IL-4, and IL-5. Effector memory T cells are predominant inthe CD8 compartment in blood, and in the human are proportionallyenriched in the lung, liver, and gut. CD8+ effector memory T cells carrylarge amounts of perforin.

The term “closed system” refers to a system that is closed to theoutside environment. Any closed system appropriate for cell culturemethods can be employed with the methods of the present invention.Closed systems include, for example, but are not limited to closedG-containers. Once a tumor segment is added to the closed system, thesystem is no opened to the outside environment until the TILs are readyto be administered to the patient.

The terms “fragmenting,” “fragment,” and “fragmented,” as used herein todescribe processes for disrupting a tumor, includes mechanicalfragmentation methods such as crushing, slicing, dividing, andmorcellating tumor tissue as well as any other method for disrupting thephysical structure of tumor tissue.

The terms “peripheral blood mononuclear cells” and “PBMCs” refers to aperipheral blood cell having a round nucleus, including lymphocytes (Tcells, B cells, NK cells) and monocytes. Preferably, the peripheralblood mononuclear cells are irradiated allogeneic peripheral bloodmononuclear cells. PBMCs are a type of antigen-presenting cell.

The term “anti-CD3 antibody” refers to an antibody or variant thereof,e.g., a monoclonal antibody and including human, humanized, chimeric ormurine antibodies which are directed against the CD3 receptor in the Tcell antigen receptor of mature T cells. Anti-CD3 antibodies includeOKT-3, also known as muromonab. Anti-CD3 antibodies also include theUHCT1 clone, also known as T3 and CD3E. Other anti-CD3 antibodiesinclude, for example, otelixizumab, teplizumab, and visilizumab.

The term “OKT-3” (also referred to herein as “OKT3”) refers to amonoclonal antibody or biosimilar or variant thereof, including human,humanized, chimeric, or murine antibodies, directed against the CD3receptor in the T cell antigen receptor of mature T cells, and includescommercially-available forms such as OKT-3 (30 ng/mL, MACS GMP CD3 pure,Miltenyi Biotech, Inc., San Diego, Calif., USA) and muromonab orvariants, conservative amino acid substitutions, glycoforms, orbiosimilars thereof. The amino acid sequences of the heavy and lightchains of muromonab are given in Table 1 (SEQ ID NO:1 and SEQ ID NO:2).A hybridoma capable of producing OKT-3 is deposited with the AmericanType Culture Collection and assigned the ATCC accession number CRL 8001.A hybridoma capable of producing OKT-3 is also deposited with EuropeanCollection of Authenticated Cell Cultures (ECACC) and assigned CatalogueNo. 86022706. Anti-CD3 antibodies also include the UHCT1 clone(commercially available from BioLegend, San Diego, Calif., USA), alsoknown as T3 and CD3E.

TABLE 1  Amino acid sequences of muromonab. IdentifierSequence (One-Letter Amino Acid Symbols) SEQ ID NO: 1QVQLQQSGAE LARPGASVKM SCKASGYTFT RYTMHWVKQR PGQGLEWIGY INPSRGYTNY 60Muromonab NQKFKDKATL TTDKSSSTAY MQLSSLTSED SAVYYCARYY DDHYCLDYWG QGTTLTVSSA 120heavy chainKTTAPSVYPL APVCGGTTGS SVTLGCLVKG YFPEPVTLTW NSGSLSSGVH TFPAVLQSDL 180YTLSSSVTVT SSTWPSQSIT CNVAHPASST KVDKKIEPRP KSCDKTHTCP PCPAPELLGG 240PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN 300STYRVVSVIT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE 360LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 420QQGNVFSCSV MHEALHNHYT QKSLSLSPGK 450 SEQ ID NO: 2QIVLTQSPAI MSASPGEKVT MTCSASSSVS YMNWYQQKSG TSPKRWIYDT SKLASGVPAH 60Muromonab FRGSGSGTSY SLTISGMEAE DAATYYCQQW SSNPFTFGSG TKLEINRADT APTVSIFPPS 120light chainSEQLTSGGAS VVCFLNNFYP KDINVKWKID GSERQNGVLN SWTDQDSKDS TYSMSSTLTL 180TKDEYERHNS YTCEATHKTS TSPIVKSFNR NEC 213

The term “IL-2” (also referred to herein as “IL2”) refers to the T cellgrowth factor known as interleukin-2, and includes all forms of IL-2including human and mammalian forms, conservative amino acidsubstitutions, glycoforms, biosimilars, and variants thereof. IL-2 isdescribed, e.g., in Nelson, J Immunol. 2004, 172, 3983-88 and Malek,Annu. Rev. Immunol. 2008, 26, 453-79, the disclosures of which areincorporated by reference herein. The amino acid sequence of recombinanthuman IL-2 suitable for use in the invention is given in Table 2 (SEQ IDNO:3). For example, the term IL-2 encompasses human, recombinant formsof IL-2 such as aldesleukin (PROLEUKIN, available commercially frommultiple suppliers in 22 million IU per single use vials), as well asthe form of recombinant IL-2 commercially supplied by CellGenix, Inc.,Portsmouth, N.H., USA (CELLGRO GMP) or ProSpec-Tany TechnoGene Ltd.,East Brunswick, N.J., USA (Cat. No. CYT-209-b) and other commercialequivalents from other vendors. Aldesleukin (des-alanyl-1, serine-125human IL-2) is a nonglycosylated human recombinant form of IL-2 with amolecular weight of approximately 15 kDa. The amino acid sequence ofaldesleukin suitable for use in the invention is given in Table 2 (SEQID NO:4). The term IL-2 also encompasses pegylated forms of IL-2, asdescribed herein, including the pegylated IL2 prodrug NKTR-214,available from Nektar Therapeutics, South San Francisco, Calif., USA.NKTR-214 and pegylated IL-2 suitable for use in the invention isdescribed in U.S. Patent Application Publication No. US 2014/0328791 A1and International Patent Application Publication No. WO 2012/065086 A1,the disclosures of which are incorporated by reference herein.Alternative forms of conjugated IL-2 suitable for use in the inventionare described in U.S. Pat. Nos. 4,766,106, 5,206,344, 5,089,261 and4902,502, the disclosures of which are incorporated by reference herein.Formulations of IL-2 suitable for use in the invention are described inU.S. Pat. No. 6,706,289, the disclosure of which is incorporated byreference herein.

TABLE 2 Amino acid sequences of interleukins. IdentifierSequence (One-Letter Amino Acid Symbols) SEQ ID NO: 3MAPTSSSTKK TQLQLEHLLL DLQMILNGIN NYKNPKLTRM LTFKFYMPKK ATELKHLQCL  60recombinantEEELKPLEEV LNLAQSKNFH LRPRDLISNI NVIVLELKGS ETTFMCEYAD ETATIVEFLN 120human IL-2RWITFCQSII STLT                                                   134(rhIL-2) SEQ ID NO: 4PTSSSTKKTQ LQLEHLLLDL QMILNGINNY KNPKLTRMLT FKFYMPKKAT ELKHLQCLEE  60AldesleukinELKPLEEVIN LAQSKNFHLR PRDLISNINV IVLELKGSET TFMCEYADET ATIVEFLNRW 120ITFSQSIIST LT                                                     132SEQ ID NO: 5MHKCDITLQE IIKTLNSLTE QKTLCTELTV TDIFAASKNT TEKETFCRAA TVLRQFYSHH  60recombinantEKDTRCLGAT AQQFHRHKQL IRFLKRLDRN LWGLAGLNSC PVKEANQSTL ENFLERLKTI 120human IL-4MREKYSKCSS                                                        130(rhIL-4) SEQ ID NO: 6MDCDIEGKDG KQYESVLMVS IDQLLDSMKE IGSNCLNNEF NFFKRHICDA NKEGMFLFRA  60recombinantARKLRQFLKM NSTGDFDLHL LKVSEGTTIL LNCTGQVKGR KPAALGEAQP TKSLEENKSL 120human IL-7KEQKKLNDLC FLKRLLQEIK TCWNKILMGT KEH                              153(rhIL-7) SEQ ID NO: 7MNWVNVISDL KKIEDLIQSM HIDATLYTES DVHPSCKVTA MKCFLLELQV ISLESGDASI  60recombinantHDTVENLIIL ANNSLSSNGN VTESGCKECE ELEEKNIKEF LQSFVHIVQM FINIS      115human IL-15 (rhIL-15) SEQ ID NO: 8MQDRHMIRMR QLIDIVDQLK NYVNDLVPEF LPAPEDVETN CEWSAFSCFQ KAQLKSANTG  60recombinantNNERIINVSI KKLKRKPPST NAGRRQKHRL TCPSCDSYEK KPPKEFLERF KSLLQKMIHQ 120human IL-21HLSSRTHGSE DS                                                     132(rhIL-21)

The term “IL-4” (also referred to herein as “IL4”) refers to thecytokine known as interleukin 4, which is produced by Th2 T cells and byeosinophils, basophils, and mast cells. IL-4 regulates thedifferentiation of naïve helper T cells (Th0 cells) to Th2 T cells.Steinke and Borish, Respir. Res. 2001, 2, 66-70. Upon activation byIL-4, Th2 T cells subsequently produce additional IL-4 in a positivefeedback loop. IL-4 also stimulates B cell proliferation and class IIMHC expression, and induces class switching to IgE and IgG₁ expressionfrom B cells. Recombinant human IL-4 suitable for use in the inventionis commercially available from multiple suppliers, includingProSpec-Tany TechnoGene Ltd., East Brunswick, N.J., USA (Cat. No.CYT-211) and ThermoFisher Scientific, Inc., Waltham, Mass., USA (humanIL-15 recombinant protein, Cat. No. Gibco CTP0043). The amino acidsequence of recombinant human IL-4 suitable for use in the invention isgiven in Table 2 (SEQ ID NO:5).

The term “IL-7” (also referred to herein as “IL7”) refers to aglycosylated tissue-derived cytokine known as interleukin 7, which maybe obtained from stromal and epithelial cells, as well as from dendriticcells. Fry and Mackall, Blood 2002, 99, 3892-904. IL-7 can stimulate thedevelopment of T cells. IL-7 binds to the IL-7 receptor, a heterodimerconsisting of IL-7 receptor alpha and common gamma chain receptor, whichin a series of signals important for T cell development within thethymus and survival within the periphery. Recombinant human IL-7suitable for use in the invention is commercially available frommultiple suppliers, including ProSpec-Tany TechnoGene Ltd., EastBrunswick, N.J., USA (Cat. No. CYT-254) and ThermoFisher Scientific,Inc., Waltham, Mass., USA (human IL-15 recombinant protein, Cat. No.Gibco PHC0071). The amino acid sequence of recombinant human IL-7suitable for use in the invention is given in Table 2 (SEQ ID NO:6).

The term “IL-15” (also referred to herein as “IL15”) refers to the Tcell growth factor known as interleukin-15, and includes all forms ofIL-2 including human and mammalian forms, conservative amino acidsubstitutions, glycoforms, biosimilars, and variants thereof. IL-15 isdescribed, e.g., in Fehniger and Caligiuri, Blood 2001, 97, 14-32, thedisclosure of which is incorporated by reference herein. IL-15 shares βand γ signaling receptor subunits with IL-2. Recombinant human IL-15 isa single, non-glycosylated polypeptide chain containing 114 amino acids(and an N-terminal methionine) with a molecular mass of 12.8 kDa.Recombinant human IL-15 is commercially available from multiplesuppliers, including ProSpec-Tany TechnoGene Ltd., East Brunswick, N.J.,USA (Cat. No. CYT-230-b) and ThermoFisher Scientific, Inc., Waltham,Mass., USA (human IL-15 recombinant protein, Cat. No. 34-8159-82). Theamino acid sequence of recombinant human IL-15 suitable for use in theinvention is given in Table 2 (SEQ ID NO:7).

The term “IL-21” (also referred to herein as “IL21”) refers to thepleiotropic cytokine protein known as interleukin-21, and includes allforms of IL-21 including human and mammalian forms, conservative aminoacid substitutions, glycoforms, biosimilars, and variants thereof. IL-21is described, e.g., in Spolski and Leonard, Nat. Rev. Drug. Disc. 2014,13, 379-95, the disclosure of which is incorporated by reference herein.IL-21 is primarily produced by natural killer T cells and activatedhuman CD4⁺ T cells. Recombinant human IL-21 is a single,non-glycosylated polypeptide chain containing 132 amino acids with amolecular mass of 15.4 kDa. Recombinant human IL-21 is commerciallyavailable from multiple suppliers, including ProSpec-Tany TechnoGeneLtd., East Brunswick, N.J., USA (Cat. No. CYT-408-b) and ThermoFisherScientific, Inc., Waltham, Mass., USA (human IL-21 recombinant protein,Cat. No. 14-8219-80). The amino acid sequence of recombinant human IL-21suitable for use in the invention is given in Table 2 (SEQ ID NO:8).

When “an anti-tumor effective amount”, “an tumor-inhibiting effectiveamount”, or “therapeutic amount” is indicated, the precise amount of thecompositions of the present invention to be administered can bedetermined by a physician with consideration of individual differencesin age, weight, tumor size, extent of infection or metastasis, andcondition of the patient (subject). It can generally be stated that apharmaceutical composition comprising the tumor infiltrating lymphocytes(e.g. secondary TILs or genetically modified cytotoxic lymphocytes)described herein may be administered at a dosage of 10⁴ to 10¹¹ cells/kgbody weight (e.g., 10⁵ to 10⁶, 10⁵ to 10¹⁰, 10⁵ to 10¹¹, 10⁶ to 10¹⁰,10⁶ to 10¹¹, 10⁷ to 10¹¹, 10⁷ to 10¹⁰, 10⁸ to 10¹¹, 10⁸ to 10¹⁰, 10⁹ to10¹¹, or 10⁹ to 10¹⁰ cells/kg body weight), including all integer valueswithin those ranges. Tumor infiltrating lymphocytes (including in somecases, genetically modified cytotoxic lymphocytes) compositions may alsobe administered multiple times at these dosages. The tumor infiltratinglymphocytes (including in some cases, genetically) can be administeredby using infusion techniques that are commonly known in immunotherapy(see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). Theoptimal dosage and treatment regime for a particular patient can readilybe determined by one skilled in the art of medicine by monitoring thepatient for signs of disease and adjusting the treatment accordingly.

The term “hematological malignancy” refers to mammalian cancers andtumors of the hematopoietic and lymphoid tissues, including but notlimited to tissues of the blood, bone marrow, lymph nodes, and lymphaticsystem. Hematological malignancies are also referred to as “liquidtumors.” Hematological malignancies include, but are not limited to,acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL),small lymphocytic lymphoma (SLL), acute myelogenous leukemia (AML),chronic myelogenous leukemia (CML), acute monocytic leukemia (AMoL),Hodgkin's lymphoma, and non-Hodgkin's lymphomas. The term “B cellhematological malignancy” refers to hematological malignancies thataffect B cells.

The term “solid tumor” refers to an abnormal mass of tissue that usuallydoes not contain cysts or liquid areas. Solid tumors may be benign ormalignant. The term “solid tumor cancer refers to malignant, neoplastic,or cancerous solid tumors. Solid tumor cancers include, but are notlimited to, sarcomas, carcinomas, and lymphomas, such as cancers of thelung, breast, prostate, colon, rectum, and bladder. The tissue structureof solid tumors includes interdependent tissue compartments includingthe parenchyma (cancer cells) and the supporting stromal cells in whichthe cancer cells are dispersed and which may provide a supportingmicroenvironment.

The term “liquid tumor” refers to an abnormal mass of cells that isfluid in nature. Liquid tumor cancers include, but are not limited to,leukemias, myelomas, and lymphomas, as well as other hematologicalmalignancies. TILs obtained from liquid tumors may also be referred toherein as marrow infiltrating lymphocytes (MILs).

The term “microenvironment,” as used herein, may refer to the solid orhematological tumor microenvironment as a whole or to an individualsubset of cells within the microenvironment. The tumor microenvironment,as used herein, refers to a complex mixture of “cells, soluble factors,signaling molecules, extracellular matrices, and mechanical cues thatpromote neoplastic transformation, support tumor growth and invasion,protect the tumor from host immunity, foster therapeutic resistance, andprovide niches for dominant metastases to thrive,” as described inSwartz, et al., Cancer Res., 2012, 72, 2473. Although tumors expressantigens that should be recognized by T cells, tumor clearance by theimmune system is rare because of immune suppression by themicroenvironment.

In an embodiment, the invention includes a method of treating a cancerwith a population of TILs, wherein a patient is pre-treated withnon-myeloablative chemotherapy prior to an infusion of TILs according tothe invention. In some embodiments, the population of TILs may beprovided wherein a patient is pre-treated with nonmyeloablativechemotherapy prior to an infusion of TILs according to the presentinvention. In an embodiment, the non-myeloablative chemotherapy iscyclophosphamide 60 mg/kg/d for 2 days (days 27 and 26 prior to TILinfusion) and fludarabine 25 mg/m2/d for 5 days (days 27 to 23 prior toTIL infusion). In an embodiment, after non-myeloablative chemotherapyand TIL infusion (at day 0) according to the invention, the patientreceives an intravenous infusion of IL-2 intravenously at 720,000 IU/kgevery 8 hours to physiologic tolerance.

Experimental findings indicate that lymphodepletion prior to adoptivetransfer of tumor-specific T lymphocytes plays a key role in enhancingtreatment efficacy by eliminating regulatory T cells and competingelements of the immune system (“cytokine sinks”). Accordingly, someembodiments of the invention utilize a lymphodepletion step (sometimesalso referred to as “immunosuppressive conditioning”) on the patientprior to the introduction of the rTILs of the invention.

The terms “co-administration,” “co-administering,” “administered incombination with,” “administering in combination with,” “simultaneous,”and “concurrent,” as used herein, encompass administration of two ormore active pharmaceutical ingredients (in a preferred embodiment of thepresent invention, for example, at least one potassium channel agonistin combination with a plurality of TILs) to a subject so that bothactive pharmaceutical ingredients and/or their metabolites are presentin the subject at the same time. Co-administration includes simultaneousadministration in separate compositions, administration at differenttimes in separate compositions, or administration in a composition inwhich two or more active pharmaceutical ingredients are present.Simultaneous administration in separate compositions and administrationin a composition in which both agents are present are preferred.

The term “effective amount” or “therapeutically effective amount” refersto that amount of a compound or combination of compounds as describedherein that is sufficient to effect the intended application including,but not limited to, disease treatment. A therapeutically effectiveamount may vary depending upon the intended application (in vitro or invivo), or the subject and disease condition being treated (e.g., theweight, age and gender of the subject), the severity of the diseasecondition, or the manner of administration. The term also applies to adose that will induce a particular response in target cells (e.g., thereduction of platelet adhesion and/or cell migration). The specific dosewill vary depending on the particular compounds chosen, the dosingregimen to be followed, whether the compound is administered incombination with other compounds, timing of administration, the tissueto which it is administered, and the physical delivery system in whichthe compound is carried.

The terms “treatment”, “treating”, “treat”, and the like, refer toobtaining a desired pharmacologic and/or physiologic effect. The effectmay be prophylactic in terms of completely or partially preventing adisease or symptom thereof and/or may be therapeutic in terms of apartial or complete cure for a disease and/or adverse effectattributable to the disease. “Treatment”, as used herein, covers anytreatment of a disease in a mammal, particularly in a human, andincludes: (a) preventing the disease from occurring in a subject whichmay be predisposed to the disease but has not yet been diagnosed ashaving it; (b) inhibiting the disease, i.e., arresting its developmentor progression; and (c) relieving the disease, i.e., causing regressionof the disease and/or relieving one or more disease symptoms.“Treatment” is also meant to encompass delivery of an agent in order toprovide for a pharmacologic effect, even in the absence of a disease orcondition. For example, “treatment” encompasses delivery of acomposition that can elicit an immune response or confer immunity in theabsence of a disease condition, e.g., in the case of a vaccine.

The term “heterologous” when used with reference to portions of anucleic acid or protein indicates that the nucleic acid or proteincomprises two or more subsequences that are not found in the samerelationship to each other in nature. For instance, the nucleic acid istypically recombinantly produced, having two or more sequences fromunrelated genes arranged to make a new functional nucleic acid, e.g., apromoter from one source and a coding region from another source, orcoding regions from different sources. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

The terms “sequence identity,” “percent identity,” and “sequence percentidentity” (or synonyms thereof, e.g., “99% identical”) in the context oftwo or more nucleic acids or polypeptides, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of nucleotides or amino acid residues that are the same, whencompared and aligned (introducing gaps, if necessary) for maximumcorrespondence, not considering any conservative amino acidsubstitutions as part of the sequence identity. The percent identity canbe measured using sequence comparison software or algorithms or byvisual inspection. Various algorithms and software are known in the artthat can be used to obtain alignments of amino acid or nucleotidesequences. Suitable programs to determine percent sequence identityinclude for example the BLAST suite of programs available from the U.S.Government's National Center for Biotechnology Information BLAST website. Comparisons between two sequences can be carried using either theBLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acidsequences, while BLASTP is used to compare amino acid sequences. ALIGN,ALIGN-2 (Genentech, South San Francisco, Calif.) or MegAlign, availablefrom DNASTAR, are additional publicly available software programs thatcan be used to align sequences. One skilled in the art can determineappropriate parameters for maximal alignment by particular alignmentsoftware. In certain embodiments, the default parameters of thealignment software are used.

As used herein, the term “variant” encompasses but is not limited toantibodies or fusion proteins which comprise an amino acid sequencewhich differs from the amino acid sequence of a reference antibody byway of one or more substitutions, deletions and/or additions at certainpositions within or adjacent to the amino acid sequence of the referenceantibody. The variant may comprise one or more conservativesubstitutions in its amino acid sequence as compared to the amino acidsequence of a reference antibody. Conservative substitutions mayinvolve, e.g., the substitution of similarly charged or uncharged aminoacids. The variant retains the ability to specifically bind to theantigen of the reference antibody. The term variant also includespegylated antibodies or proteins.

By “tumor infiltrating lymphocytes” or “TILs” herein is meant apopulation of cells originally obtained as white blood cells that haveleft the bloodstream of a subject and migrated into a tumor. TILsinclude, but are not limited to, CD8⁺ cytotoxic T cells (lymphocytes),Th1 and Th17 CD4⁺ T cells, natural killer cells, dendritic cells and M1macrophages. TILs include both primary and secondary TILs. “PrimaryTILs” are those that are obtained from patient tissue samples asoutlined herein (sometimes referred to as “freshly harvested”), and“secondary TILs” are any TIL cell populations that have been expanded orproliferated as discussed herein, including, but not limited to bulkTILs, expanded TILs (“REP TILs”) as well as “reREP TILs” as discussedherein. reREP TILs can include for example second expansion TILs orsecond additional expansion TILs (such as, for example, those describedin Step D of FIG. 9, including TILs referred to as reREP TILs).

TILs can generally be defined either biochemically, using cell surfacemarkers, or functionally, by their ability to infiltrate tumors andeffect treatment. TILs can be generally categorized by expressing one ormore of the following biomarkers: CD4, CD8, TCR αβ, CD27, CD28, CD56,CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally, and alternatively,TILs can be functionally defined by their ability to infiltrate solidtumors upon reintroduction into a patient. TILS may further becharacterized by potency—for example, TILS may be considered potent if,for example, interferon (IFN) release is greater than about 50 pg/mL,greater than about 100 pg/mL, greater than about 150 pg/mL, or greaterthan about 200 pg/mL.

The terms “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable excipient” are intended to include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and inert ingredients. The useof such pharmaceutically acceptable carriers or pharmaceuticallyacceptable excipients for active pharmaceutical ingredients is wellknown in the art. Except insofar as any conventional pharmaceuticallyacceptable carrier or pharmaceutically acceptable excipient isincompatible with the active pharmaceutical ingredient, its use in thetherapeutic compositions of the invention is contemplated. Additionalactive pharmaceutical ingredients, such as other drugs, can also beincorporated into the described compositions and methods.

The terms “about” and “approximately” mean within a statisticallymeaningful range of a value. Such a range can be within an order ofmagnitude, preferably within 50%, more preferably within 20%, morepreferably still within 10%, and even more preferably within 5% of agiven value or range. The allowable variation encompassed by the terms“about” or “approximately” depends on the particular system under study,and can be readily appreciated by one of ordinary skill in the art.Moreover, as used herein, the terms “about” and “approximately” meanthat dimensions, sizes, formulations, parameters, shapes and otherquantities and characteristics are not and need not be exact, but may beapproximate and/or larger or smaller, as desired, reflecting tolerances,conversion factors, rounding off, measurement error and the like, andother factors known to those of skill in the art. In general, adimension, size, formulation, parameter, shape or other quantity orcharacteristic is “about” or “approximate” whether or not expresslystated to be such. It is noted that embodiments of very different sizes,shapes and dimensions may employ the described arrangements.

The transitional terms “comprising,” “consisting essentially of,” and“consisting of,” when used in the appended claims, in original andamended form, define the claim scope with respect to what unrecitedadditional claim elements or steps, if any, are excluded from the scopeof the claim(s). The term “comprising” is intended to be inclusive oropen-ended and does not exclude any additional, unrecited element,method, step or material. The term “consisting of” excludes any element,step or material other than those specified in the claim and, in thelatter instance, impurities ordinary associated with the specifiedmaterial(s). The term “consisting essentially of” limits the scope of aclaim to the specified elements, steps or material(s) and those that donot materially affect the basic and novel characteristic(s) of theclaimed invention. All compositions, methods, and kits described hereinthat embody the present invention can, in alternate embodiments, be morespecifically defined by any of the transitional terms “comprising,”“consisting essentially of,” and “consisting of”

III. Gene-Editing Processes

A. Overview: TIL Expansion+Gene-Editing

Embodiments of the present invention are directed to methods forexpanding TIL populations, the methods comprising one or more steps ofgene-editing at least a portion of the TILs in order to enhance theirtherapeutic effect. As used herein, “gene-editing,” “gene editing,” and“genome editing” refer to a type of genetic modification in which DNA ispermanently modified in the genome of a cell, e.g., DNA is inserted,deleted, modified or replaced within the cell's genome. In someembodiments, gene-editing causes the expression of a DNA sequence to besilenced (sometimes referred to as a gene knockout) or inhibited/reduced(sometimes referred to as a gene knockdown). In other embodiments,gene-editing causes the expression of a DNA sequence to be enhanced(e.g., by causing over-expression). In accordance with embodiments ofthe present invention, gene-editing technology is used to enhance theeffectiveness of a therapeutic population of TILs.

A method for expanding tumor infiltrating lymphocytes (TILs) into atherapeutic population of TILs may be carried out in accordance with anyembodiment of the methods described herein (e.g., an exemplary TILexpansion method known as process 2A is described below), wherein themethod further comprises gene-editing at least a portion of the TILs.According to additional embodiments, a method for expanding TILs into atherapeutic population of TILs is carried out in accordance with anyembodiment of the methods described in PCT/US2017/058610,PCT/US2018/012605, or PCT/US2018/012633, which are incorporated byreference herein in their entireties, wherein the method furthercomprises gene-editing at least a portion of the TILs. Thus, anembodiment of the present invention provides a therapeutic population ofTILs that has been expanded in accordance with any embodiment describedherein, wherein at least a portion of the therapeutic population hasbeen gene-edited, e.g., at least a portion of the therapeutic populationof TILs that is transferred to the infusion bag is permanentlygene-edited.

B. Timing of Gene-Editing During TIL Expansion

According to one embodiment, a method for expanding tumor infiltratinglymphocytes (TILs) into a therapeutic population of TILs comprises:

(a) obtaining a first population of TILs from a tumor resected from apatient by processing a tumor sample obtained from the patient intomultiple tumor fragments;

(b) adding the tumor fragments into a closed system;

(c) performing a first expansion by culturing the first population ofTILs in a cell culture medium comprising IL-2, and optionally OKT-3(e.g., OKT-3 may be present in the culture medium beginning on the startdate of the expansion process), to produce a second population of TILs,wherein the first expansion is performed in a closed container providinga first gas-permeable surface area, wherein the first expansion isperformed for about 3-14 days to obtain the second population of TILs,wherein the second population of TILs is at least 50-fold greater innumber than the first population of TILs, and wherein the transitionfrom step (b) to step (c) occurs without opening the system;

(d) performing a second expansion by supplementing the cell culturemedium of the second population of TILs with additional IL-2, optionallyOKT-3, and antigen presenting cells (APCs), to produce a thirdpopulation of TILs, wherein the second expansion is performed for about7-14 days to obtain the third population of TILs, wherein the thirdpopulation of TILs is a therapeutic population of TILs, wherein thesecond expansion is performed in a closed container providing a secondgas-permeable surface area, and wherein the transition from step (c) tostep (d) occurs without opening the system;

(e) harvesting the therapeutic population of TILs obtained from step(d), wherein the transition from step (d) to step (e) occurs withoutopening the system;

(f) transferring the harvested TIL population from step (e) to aninfusion bag, wherein the transfer from step (e) to (f) occurs withoutopening the system; and

(g) at any time during the method, gene-editing at least a portion ofthe TILs.

As stated in step (g) of the embodiment described above, thegene-editing process may be carried out at any time during the TILexpansion method, which means that the gene editing may be carried outon TILs before, during, or after any of the steps in the expansionmethod; for example, during any of steps (a)-(f) outlined in the methodabove, or before or after any of steps (a)-(f) outlined in the methodabove. According to certain embodiments, TILs are collected during theexpansion method (e.g., the expansion method is “paused” for at least aportion of the TILs), and the collected TILs are subjected to agene-editing process, and, in some cases, subsequently reintroduced backinto the expansion method (e.g., back into the culture medium) tocontinue the expansion process, so that at least a portion of thetherapeutic population of TILs that are eventually transferred to theinfusion bag are permanently gene-edited. In an embodiment, thegene-editing process may be carried out before expansion by activatingTILs, performing a gene-editing step on the activated TILs, andexpanding the gene-edited TILs according to the processes describedherein.

It should be noted that alternative embodiments of the expansion processmay differ from the method shown above; e.g., alternative embodimentsmay not have the same steps (a)-(g), or may have a different number ofsteps. Regardless of the specific embodiment, the gene-editing processmay be carried out at any time during the TIL expansion method. Forexample, alternative embodiments may include more than two expansions,and it is possible that gene-editing may be conducted on the TILs duringa third or fourth expansion, etc.

According to one embodiment, the gene-editing process is carried out onTILs from one or more of the first population, the second population,and the third population. For example, gene-editing may be carried outon the first population of TILs, or on a portion of TILs collected fromthe first population, and following the gene-editing process those TILsmay subsequently be placed back into the expansion process (e.g., backinto the culture medium). Alternatively, gene-editing may be carried outon TILs from the second or third population, or on a portion of TILscollected from the second or third population, respectively, andfollowing the gene-editing process those TILs may subsequently be placedback into the expansion process (e.g., back into the culture medium).According to another embodiment, gene-editing is performed while theTILs are still in the culture medium and while the expansion is beingcarried out, i.e., they are not necessarily “removed” from the expansionin order to conduct gene-editing.

According to another embodiment, the gene-editing process is carried outon TILs from the first expansion, or TILs from the second expansion, orboth. For example, during the first expansion or second expansion,gene-editing may be carried out on TILs that are collected from theculture medium, and following the gene-editing process those TILs maysubsequently be placed back into the expansion method, e.g., byreintroducing them back into the culture medium.

According to another embodiment, the gene-editing process is carried outon at least a portion of the TILs after the first expansion and beforethe second expansion. For example, after the first expansion,gene-editing may be carried out on TILs that are collected from theculture medium, and following the gene-editing process those TILs maysubsequently be placed back into the expansion method, e.g., byreintroducing them back into the culture medium for the secondexpansion.

According to alternative embodiments, the gene-editing process iscarried out before step (c) (e.g., before, during, or after any of steps(a)-(b)), before step (d) (e.g., before, during, or after any of steps(a)-(c)), before step (e) (e.g., before, during, or after any of steps(a)-(d)), or before step (f) (e.g., before, during, or after any ofsteps (a)-(e)).

It should be noted with regard to OKT-3, according to certainembodiments, that the cell culture medium may comprise OKT-3 beginningon the start day (Day 0), or on Day 1 of the first expansion, such thatthe gene-editing is carried out on TILs after they have been exposed toOKT-3 in the cell culture medium on Day 0 and/or Day 1. According toanother embodiment, the cell culture medium comprises OKT-3 during thefirst expansion and/or during the second expansion, and the gene-editingis carried out before the OKT-3 is introduced into the cell culturemedium. Alternatively, the cell culture medium may comprise OKT-3 duringthe first expansion and/or during the second expansion, and thegene-editing is carried out after the OKT-3 is introduced into the cellculture medium.

It should also be noted with regard to a 4-1BB agonist, according tocertain embodiments, that the cell culture medium may comprise a 4-1BBagonist beginning on the start day (Day 0), or on Day 1 of the firstexpansion, such that the gene-editing is carried out on TILs after theyhave been exposed to a 4-1BB agonist in the cell culture medium on Day 0and/or Day 1. According to another embodiment, the cell culture mediumcomprises a 4-1BB agonist during the first expansion and/or during thesecond expansion, and the gene-editing is carried out before the 4-1BBagonist is introduced into the cell culture medium. Alternatively, thecell culture medium may comprise a 4-1BB agonist during the firstexpansion and/or during the second expansion, and the gene-editing iscarried out after the 4-1BB agonist is introduced into the cell culturemedium.

It should also be noted with regard to IL-2, according to certainembodiments, that the cell culture medium may comprise IL-2 beginning onthe start day (Day 0), or on Day 1 of the first expansion, such that thegene-editing is carried out on TILs after they have been exposed to IL-2in the cell culture medium on Day 0 and/or Day 1. According to anotherembodiment, the cell culture medium comprises IL-2 during the firstexpansion and/or during the second expansion, and the gene-editing iscarried out before the IL-2 is introduced into the cell culture medium.Alternatively, the cell culture medium may comprise IL-2 during thefirst expansion and/or during the second expansion, and the gene-editingis carried out after the IL-2 is introduced into the cell culturemedium.

As discussed above, one or more of OKT-3, 4-1BB agonist and IL-2 may beincluded in the cell culture medium beginning on Day 0 or Day 1 of thefirst expansion. According to one embodiment, OKT-3 is included in thecell culture medium beginning on Day 0 or Day 1 of the first expansion,and/or a 4-1BB agonist is included in the cell culture medium beginningon Day 0 or Day 1 of the first expansion, and/or IL-2 is included in thecell culture medium beginning on Day 0 or Day 1 of the first expansion.According to an example, the cell culture medium comprises OKT-3 and a4-1BB agonist beginning on Day 0 or Day 1 of the first expansion.According to another example, the cell culture medium comprises OKT-3, a4-1BB agonist and IL-2 beginning on Day 0 or Day 1 of the firstexpansion. Of course, one or more of OKT-3, 4-1BB agonist and IL-2 maybe added to the cell culture medium at one or more additional timepoints during the expansion process, as set forth in various embodimentsdescribed herein.

According to one embodiment, a method for expanding tumor infiltratinglymphocytes (TILs) into a therapeutic population of TILs comprises:

(a) obtaining a first population of TILs from a tumor resected from apatient by processing a tumor sample obtained from the patient intomultiple tumor fragments;

(b) adding the tumor fragments into a closed system;

(c) performing a first expansion by culturing the first population ofTILs in a cell culture medium comprising IL-2 and optionally comprisinga 4-1BB agonist antibody for about 2 to 5 days;

(d) adding OKT-3, to produce a second population of TILs, wherein thefirst expansion is performed in a closed container providing a firstgas-permeable surface area, wherein the first expansion is performed forabout 1 to 3 days to obtain the second population of TILs, wherein thesecond population of TILs is at least 50-fold greater in number than thefirst population of TILs, and wherein the transition from step (c) tostep (d) occurs without opening the system;

(e) performing a sterile electroporation step on the second populationof TILs, wherein the sterile electroporation step mediates the transferof at least one gene editor;

(f) resting the second population of TILs for about 1 day;

(g) performing a second expansion by supplementing the cell culturemedium of the second population of TILs with additional IL-2, optionallyOKT-3 antibody, optionally an OX40 antibody, and antigen presentingcells (APCs), to produce a third population of TILs, wherein the secondexpansion is performed for about 7 to 11 days to obtain the thirdpopulation of TILs, wherein the second expansion is performed in aclosed container providing a second gas-permeable surface area, andwherein the transition from step (f) to step (g) occurs without openingthe system;

(h) harvesting the therapeutic population of TILs obtained from step (g)to provide a harvested TIL population, wherein the transition from step(g) to step (h) occurs without opening the system, wherein the harvestedpopulation of TILs is a therapeutic population of TILs;

(i) transferring the harvested TIL population to an infusion bag,wherein the transfer from step (h) to (i) occurs without opening thesystem; and

(j) cryopreserving the harvested TIL population using adimethylsulfoxide-based cryopreservation medium.

According to one embodiment, the foregoing method may be used to providean autologous harvested TIL population for the treatment of a humansubject with cancer.

C. Immune Checkpoints

According to particular embodiments of the present invention, a TILpopulation is gene-edited by genetically modifying one or more immunecheckpoint genes in the TIL population. Stated another way, a DNAsequence within the TIL that encodes one or more of the TIL's immunecheckpoints is permanently modified, e.g., inserted, deleted orreplaced, in the TIL's genome. Immune checkpoints are moleculesexpressed by lymphocytes that regulate an immune response via inhibitoryor stimulatory pathways. In the case of cancer, immune checkpointpathways are often activated to inhibit the anti-tumor response, i.e.,the expression of certain immune checkpoints by malignant cells inhibitsthe anti-tumor immunity and favors the growth of cancer cells. See,e.g., Marin-Acevedo et al., Journal of Hematology & Oncology (2018)11:39. Thus, certain inhibitory checkpoint molecules serve as targetsfor immunotherapies of the present invention. According to particularembodiments, TILs are gene-edited to block or stimulate certain immunecheckpoint pathways and thereby enhance the body's immunologicalactivity against tumors.

As used herein, an immune checkpoint gene comprises a DNA sequenceencoding an immune checkpoint molecule. According to particularembodiments of the present invention, gene-editing TILs during the TILexpansion method causes expression of one or more immune checkpointgenes to be silenced or reduced in at least a portion of the therapeuticpopulation of TILs. For example, gene-editing may cause the expressionof an inhibitory receptor, such as PD-1 or CTLA-4, to be silenced orreduced in order to enhance an immune reaction.

The most broadly studied checkpoints include programmed cell deathreceptor-1 (PD-1) and cytotoxic T lymphocyte-associated molecule-4(CTLA-4), which are inhibitory receptors on immune cells that inhibitkey effector functions (e.g., activation, proliferation, cytokinerelease, cytoxicity, etc.) when they interact with an inhibitory ligand.Numerous checkpoint molecules, in addition to PD-1 and CTLA-4, haveemerged as potential targets for immunotherapy, as discussed in moredetail below.

Non-limiting examples of immune checkpoint genes that may be silenced orinhibited by permanently gene-editing TILs of the present inventioninclude PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B,PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, BAFF (BR3),CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A,CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4,SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4,CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, andGUCY1B3. For example, immune checkpoint genes that may be silenced orinhibited in TILs of the present invention may be selected from thegroup comprising PD-1, CTLA-4, LAG-3, TIM-3, Cish, TGFβ, and PKA. BAFF(BR3) is described in Bloom, et al., J Immunother., 2018, in press.According to another example, immune checkpoint genes that may besilenced or inhibited in TILs of the present invention may be selectedfrom the group comprising PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH,TGFβR2, PRA, CBLB, BAFF (BR3), and combinations thereof.

According to one embodiment, a method for expanding tumor infiltratinglymphocytes (TILs) into a therapeutic population of TILs comprises:

(a) obtaining a first population of TILs from a tumor resected from apatient by processing a tumor sample obtained from the patient intomultiple tumor fragments;

(b) adding the tumor fragments into a closed system;

(c) performing a first expansion by culturing the first population ofTILs in a cell culture medium comprising IL-2 and optionally comprisinga 4-1BB agonist antibody for about 2 to 5 days;

(d) adding OKT-3, to produce a second population of TILs, wherein thefirst expansion is performed in a closed container providing a firstgas-permeable surface area, wherein the first expansion is performed forabout 1 to 3 days to obtain the second population of TILs, wherein thesecond population of TILs is at least 50-fold greater in number than thefirst population of TILs, and wherein the transition from step (c) tostep (d) occurs without opening the system;

(e) performing a sterile electroporation step on the second populationof TILs, wherein the sterile electroporation step mediates the transferof at least one gene editor;

(f) resting the second population of TILs for about 1 day;

(g) performing a second expansion by supplementing the cell culturemedium of the second population of TILs with additional IL-2, optionallyOKT-3 antibody, optionally an OX40 antibody, and antigen presentingcells (APCs), to produce a third population of TILs, wherein the secondexpansion is performed for about 7 to 11 days to obtain the thirdpopulation of TILs, wherein the second expansion is performed in aclosed container providing a second gas-permeable surface area, andwherein the transition from step (f) to step (g) occurs without openingthe system;

(h) harvesting the therapeutic population of TILs obtained from step (g)to provide a harvested TIL population, wherein the transition from step(g) to step (h) occurs without opening the system, wherein the harvestedpopulation of TILs is a therapeutic population of TILs;

(i) transferring the harvested TIL population to an infusion bag,wherein the transfer from step (h) to (i) occurs without opening thesystem; and

(j) cryopreserving the harvested TIL population using adimethylsulfoxide-based cryopreservation medium,

wherein the electroporation step comprises the delivery of a ClusteredRegularly Interspersed Short Palindromic Repeat (CRISPR) system, aTranscription Activator-Like Effector (TALE) system, or a zinc fingersystem for inhibiting the expression of a molecule selected from thegroup consisting of PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGFβR2,PRA, CBLB, BAFF (BR3), and combinations thereof.

1. PD-1

One of the most studied targets for the induction of checkpoint blockadeis the programmed death receptor (PD1 or PD-1, also known as PDCD1), amember of the CD28 super family of T-cell regulators. Its ligands, PD-L1and PD-L2, are expressed on a variety of tumor cells, includingmelanoma. The interaction of PD-1 with PD-L1 inhibits T-cell effectorfunction, results in T-cell exhaustion in the setting of chronicstimulation, and induces T-cell apoptosis in the tumor microenvironment.PD1 may also play a role in tumor-specific escape from immunesurveillance.

According to particular embodiments, expression of PD1 in TILs issilenced or reduced in accordance with compositions and methods of thepresent invention. For example, a method for expanding tumorinfiltrating lymphocytes (TILs) into a therapeutic population of TILsmay be carried out in accordance with any embodiment of the methodsdescribed herein (e.g., process 2A or the methods shown in FIGS. 20 and21), wherein the method comprises gene-editing at least a portion of theTILs by silencing or repressing the expression of PD1. As described inmore detail below, the gene-editing process may involve the use of aprogrammable nuclease that mediates the generation of a double-strand orsingle-strand break at an immune checkpoint gene, such as PD1. Forexample, a CRISPR method, a TALE method, or a zinc finger method may beused to silence or reduce the expression of PD1 in the TILs.

2. CTLA-4

CTLA-4 expression is induced upon T-cell activation on activatedT-cells, and competes for binding with the antigen presenting cellactivating antigens CD80 and CD86. Interaction of CTLA-4 with CD80 orCD86 causes T-cell inhibition and serves to maintain balance of theimmune response. However, inhibition of the CTLA-4 interaction with CD80or CD86 may prolong T-cell activation and thus increase the level ofimmune response to a cancer antigen.

According to particular embodiments, expression of CTLA-4 in TILs issilenced or reduced in accordance with compositions and methods of thepresent invention. For example, a method for expanding tumorinfiltrating lymphocytes (TILs) into a therapeutic population of TILsmay be carried out in accordance with any embodiment of the methodsdescribed herein (e.g., process 2A or the methods shown in FIGS. 20 and21), wherein the method comprises gene-editing at least a portion of theTILs by silencing or repressing the expression of CTLA-4. As describedin more detail below, the gene-editing process may comprise the use of aprogrammable nuclease that mediates the generation of a double-strand orsingle-strand break at an immune checkpoint gene, such as CTLA-4. Forexample, a CRISPR method, a TALE method, or a zinc finger method may beused to silence or repress the expression of CTLA-4 in the TILs.

3. LAG-3

Lymphocyte activation gene-3 (LAG-3, CD223) is expressed by T cells andnatural killer (NK) cells after major histocompatibility complex (MHC)class II ligation. Although its mechanism remains unclear, itsmodulation causes a negative regulatory effect over T cell function,preventing tissue damage and autoimmunity. LAG-3 and PD-1 are frequentlyco-expressed and upregulated on TILs, leading to immune exhaustion andtumor growth. Thus, LAG-3 blockade improves anti-tumor responses. See,e.g., Marin-Acevedo et al., Journal of Hematology & Oncology (2018)11:39.

According to particular embodiments, expression of LAG-3 in TILs issilenced or reduced in accordance with compositions and methods of thepresent invention. For example, a method for expanding tumorinfiltrating lymphocytes (TILs) into a therapeutic population of TILsmay be carried out in accordance with any embodiment of the methodsdescribed herein (e.g., process 2A or the methods shown in FIGS. 20 and21), wherein the method comprises gene-editing at least a portion of theTILs by silencing or repressing the expression of LAG-3. As described inmore detail below, the gene-editing process may comprise the use of aprogrammable nuclease that mediates the generation of a double-strand orsingle-strand break at an immune checkpoint gene, such as LAG-3.According to particular embodiments, a CRISPR method, a TALE method, ora zinc finger method may be used to silence or repress the expression ofLAG-3 in the TILs.

4. TIM-3

T cell immunoglobulin-3 (TIM-3) is a direct negative regulator of Tcells and is expressed on NK cells and macrophages. TIM-3 indirectlypromotes immunosuppression by inducing expansion of myeloid-derivedsuppressor cells (MDSCs). Its levels have been found to be particularlyelevated on dysfunctional and exhausted T-cells, suggesting an importantrole in malignancy.

According to particular embodiments, expression of TIM-3 in TILs issilenced or reduced in accordance with compositions and methods of thepresent invention. For example, a method for expanding tumorinfiltrating lymphocytes (TILs) into a therapeutic population of TILsmay be carried out in accordance with any embodiment of the methodsdescribed herein (e.g., process 2A or the methods shown in FIGS. 20 and21), wherein the method comprises gene-editing at least a portion of theTILs by silencing or repressing the expression of TIM-3. As described inmore detail below, the gene-editing process may comprise the use of aprogrammable nuclease that mediates the generation of a double-strand orsingle-strand break at an immune checkpoint gene, such as TIM-3. Forexample, a CRISPR method, a TALE method, or a zinc finger method may beused to silence or repress the expression of TIM-3 in the TILs.

5. Cish

Cish, a member of the suppressor of cytokine signaling (SOCS) family, isinduced by TCR stimulation in CD8+ T cells and inhibits their functionalavidity against tumors. Genetic deletion of Cish in CD8+ T cells mayenhance their expansion, functional avidity, and cytokinepolyfunctionality, resulting in pronounced and durable regression ofestablished tumors. See, e.g., Palmer et al., Journal of ExperimentalMedicine, 212 (12): 2095 (2015).

According to particular embodiments, expression of Cish in TILs issilenced or reduced in accordance with compositions and methods of thepresent invention. For example, a method for expanding tumorinfiltrating lymphocytes (TILs) into a therapeutic population of TILsmay be carried out in accordance with any embodiment of the methodsdescribed herein (e.g., process 2A or the methods shown in FIGS. 20 and21), wherein the method comprises gene-editing at least a portion of theTILs by silencing or repressing the expression of Cish. As described inmore detail below, the gene-editing process may comprise the use of aprogrammable nuclease that mediates the generation of a double-strand orsingle-strand break at an immune checkpoint gene, such as Cish. Forexample, a CRISPR method, a TALE method, or a zinc finger method may beused to silence or repress the expression of Cish in the TILs.

6. TGFβ

The TGFβ signaling pathway has multiple functions in regulating cellgrowth, differentiation, apoptosis, motility and invasion, extracellularmatrix production, angiogenesis, and immune response. TGFβ signalingderegulation is frequent in tumors and has crucial roles in tumorinitiation, development and metastasis. At the microenvironment level,the TGFβ pathway contributes to generate a favorable microenvironmentfor tumor growth and metastasis throughout carcinogenesis. See, e.g.,Neuzillet et al., Pharmacology & Therapeutics, Vol. 147, pp. 22-31(2015).

According to particular embodiments, expression of TGFβ in TILs issilenced or reduced in accordance with compositions and methods of thepresent invention. For example, a method for expanding tumorinfiltrating lymphocytes (TILs) into a therapeutic population of TILsmay be carried out in accordance with any embodiment of the methodsdescribed herein (e.g., process 2A or the methods shown in FIGS. 20 and21), wherein the method comprises gene-editing at least a portion of theTILs by silencing or reducing the expression of TGFβ. As described inmore detail below, the gene-editing process may comprise the use of aprogrammable nuclease that mediates the generation of a double-strand orsingle-strand break at an immune checkpoint gene, such as TGFβ. Forexample, a CRISPR method, a TALE method, or a zinc finger method may beused to silence or repress the expression of TGFβ in the TILs.

In some embodiments, TGFβR2 (TGF beta receptor 2) may be suppressed bysilencing TGFβR2 using a CRISPR/Cas9 system or by using a TGFβR2dominant negative extracellular trap, using methods known in the art.

7. PKA

Protein Kinase A (PKA) is a well-known member of the serine-threonineprotein kinase superfamily. PKA, also known as cAMP-dependent proteinkinase, is a multi-unit protein kinase that mediates signal transductionof G-protein coupled receptors through its activation upon cAMP binding.It is involved in the control of a wide variety of cellular processesfrom metabolism to ion channel activation, cell growth anddifferentiation, gene expression and apoptosis. Importantly, PKA hasbeen implicated in the initiation and progression of many tumors. See,e.g., Sapio et al., EXCLI Journal; 2014; 13: 843-855.

According to particular embodiments, expression of PKA in TILs issilenced or reduced in accordance with compositions and methods of thepresent invention. For example, a method for expanding tumorinfiltrating lymphocytes (TILs) into a therapeutic population of TILsmay be carried out in accordance with any embodiment of the methodsdescribed herein (e.g., process 2A or the methods shown in FIGS. 20 and21), wherein the method comprises gene-editing at least a portion of theTILs by silencing or repressing the expression of PKA. As described inmore detail below, the gene-editing process may comprise the use of aprogrammable nuclease that mediates the generation of a double-strand orsingle-strand break at an immune checkpoint gene, such as PKA. Forexample, a CRISPR method, a TALE method, or a zinc finger method may beused to silence or repress the expression of PKA in the TILs.

8. CBLB

CBLB (or CBL-B) is a E3 ubiquitin-protein ligase and is a negativeregulator of T cell activation. Bachmaier, et al., Nature, 2000, 403,211-216; Wallner, et al., Clin. Dev. Immunol. 2012, 692639.

According to particular embodiments, expression of CBLB in TILs issilenced or reduced in accordance with compositions and methods of thepresent invention. For example, a method for expanding tumorinfiltrating lymphocytes (TILs) into a therapeutic population of TILsmay be carried out in accordance with any embodiment of the methodsdescribed herein (e.g., process 2A or the methods shown in FIGS. 20 and21), wherein the method comprises gene-editing at least a portion of theTILs by silencing or repressing the expression of CBLB. As described inmore detail below, the gene-editing process may comprise the use of aprogrammable nuclease that mediates the generation of a double-strand orsingle-strand break at an immune checkpoint gene, such as CBLB. Forexample, a CRISPR method, a TALE method, or a zinc finger method may beused to silence or repress the expression of PKA in the TILs. In someembodiments, CBLB is silenced using a TALEN knockout. In someembodiments, CBLB is silenced using a TALE-KRAB transcriptionalinhibitor knock in. More details on these methods can be found inBoettcher and McManus, Mol Cell Review, 2015, 58, 575-585.

9. TIGIT

T-cell immunoreceptor with Ig and ITIM (immunoreceptor tyrosine-basedinhibitory motif) domain or TIGIT is a transmembrane glycoproteinreceptor with an Ig-like V-type domain and an ITIM in its cytoplasmicdomain. Khalil, et al., Advances in Cancer Research, 2015, 128, 1-68;Yu, et al., Nature Immunology, 2009, Vol. 10, No. 1, 48-57.

TIGIT is expressed by some T cells and Natural Killer Cells.Additionally, TIGIT has been shown to be overexpressed onantigen-specific CD8+ T cells and CD8+ TILs, particularly fromindividuals with melanoma. Studies have shown that the TIGIT pathwaycontributes to tumor immune evasion and TIGIT inhibition has been shownto increase T-cell activation and proliferation in response topolyclonal and antigen-specific stimulation. Khalil, et al., Advances inCancer Research, 2015, 128, 1-68. Further, coblockade of TIGIT witheither PD-1 or TIM3 has shown synergistic effects against solid tumorsin mouse models. Id.; see also Kurtulus, et al., The Journal of ClinicalInvestigation, 2015, Vol. 125, No. 11, 4053-4062.

According to particular embodiments, expression of TIGIT in TILs issilenced or reduced in accordance with compositions and methods of thepresent invention. For example, a method for expanding tumorinfiltrating lymphocytes (TILs) into a therapeutic population of TILsmay be carried out in accordance with any embodiment of the methodsdescribed herein (e.g., process 2A or the methods shown in FIGS. 20 and21), wherein the method comprises gene-editing at least a portion of theTILs by silencing or repressing the expression of TIGIT. As described inmore detail below, the gene-editing process may comprise the use of aprogrammable nuclease that mediates the generation of a double-strand orsingle-strand break at an immune checkpoint gene, such as TIGIT. Forexample, a CRISPR method, a TALE method, or a zinc finger method may beused to silence or repress the expression of TIGIT in the TILs.

D. Overexpression of Co-Stimulatory Receptors or Adhesion Molecules

According to additional embodiments, gene-editing TILs during the TILexpansion method causes expression of one or more immune checkpointgenes to be enhanced in at least a portion of the therapeutic populationof TILs. For example, gene-editing may cause the expression of astimulatory receptor to be enhanced, which means that it isoverexpressed as compared to the expression of a stimulatory receptorthat has not been genetically modified. Non-limiting examples of immunecheckpoint genes that may exhibit enhanced expression by permanentlygene-editing TILs of the present invention include certain chemokinereceptors and interleukins, such as CCR2, CCR4, CCR5, CXCR2, CXCR3,CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-21, the NOTCH 1/2intracellular domain (ICD), and/or the NOTCH ligand mDLL1.

1. CCRs

For adoptive T cell immunotherapy to be effective, T cells need to betrafficked properly into tumors by chemokines. A match betweenchemokines secreted by tumor cells, chemokines present in the periphery,and chemokine receptors expressed by T cells is important for successfultrafficking of T cells into a tumor bed.

According to particular embodiments, gene-editing methods of the presentinvention may be used to increase the expression of certain chemokinereceptors in the TILs, such as one or more of CCR2, CCR4, CCR5, CXCR2,CXCR3 and CX3CR1. Over-expression of CCRs may help promote effectorfunction and proliferation of TILs following adoptive transfer.

According to particular embodiments, expression of one or more of CCR2,CCR4, CCR5, CXCR2, CXCR3 and CX3CR1 in TILs is enhanced in accordancewith compositions and methods of the present invention. For example, amethod for expanding tumor infiltrating lymphocytes (TILs) into atherapeutic population of TILs may be carried out in accordance with anyembodiment of the methods described herein (e.g., process 2A or themethods shown in FIGS. 20 and 21), wherein the method comprisesgene-editing at least a portion of the TILs by enhancing the expressionof one or more of CCR2, CCR4, CCR5, CXCR2, CXCR3 and CX3CR1. Asdescribed in more detail below, the gene-editing process may comprisethe use of a programmable nuclease that mediates the generation of adouble-strand or single-strand break at a chemokine receptor gene. Forexample, a CRISPR method, a TALE method, or a zinc finger method may beused to enhance the expression of certain chemokine receptors in theTILs.

In an embodiment, CCR4 and/or CCR5 adhesion molecules are inserted intoa TIL population using a gamma-retroviral or lentiviral method asdescribed herein. In an embodiment, CXCR2 adhesion molecule are insertedinto a TIL population using a gamma-retroviral or lentiviral method asdescribed in Forget, et al., Frontiers Immunology 2017, 8, 908 or Peng,et al., Clin. Cancer Res. 2010, 16, 5458, the disclosures of which areincorporated by reference herein.

According to one embodiment, a method for expanding tumor infiltratinglymphocytes (TILs) into a therapeutic population of TILs comprises:

(a) obtaining a first population of TILs from a tumor resected from apatient by processing a tumor sample obtained from the patient intomultiple tumor fragments;

(b) adding the tumor fragments into a closed system;

(c) performing a first expansion by culturing the first population ofTILs in a cell culture medium comprising IL-2 and optionally comprisinga 4-1BB agonist antibody for about 2 to 5 days;

(d) adding OKT-3, to produce a second population of TILs, wherein thefirst expansion is performed in a closed container providing a firstgas-permeable surface area, wherein the first expansion is performed forabout 1 to 3 days to obtain the second population of TILs, wherein thesecond population of TILs is at least 50-fold greater in number than thefirst population of TILs, and wherein the transition from step (c) tostep (d) occurs without opening the system;

(e) performing a sterile electroporation step on the second populationof TILs, wherein the sterile electroporation step mediates the transferof at least one gene editor;

(f) resting the second population of TILs for about 1 day;

(g) performing a second expansion by supplementing the cell culturemedium of the second population of TILs with additional IL-2, optionallyOKT-3 antibody, optionally an OX40 antibody, and antigen presentingcells (APCs), to produce a third population of TILs, wherein the secondexpansion is performed for about 7 to 11 days to obtain the thirdpopulation of TILs, wherein the second expansion is performed in aclosed container providing a second gas-permeable surface area, andwherein the transition from step (f) to step (g) occurs without openingthe system;

(h) harvesting the therapeutic population of TILs obtained from step (g)to provide a harvested TIL population, wherein the transition from step(g) to step (h) occurs without opening the system, wherein the harvestedpopulation of TILs is a therapeutic population of TILs;

(i) transferring the harvested TIL population to an infusion bag,wherein the transfer from step (h) to (i) occurs without opening thesystem; and

(j) cryopreserving the harvested TIL population using adimethylsulfoxide-based cryopreservation medium,

wherein the electroporation step comprises the delivery of a ClusteredRegularly Interspersed Short Palindromic Repeat (CRISPR) system, aTranscription Activator-Like Effector (TALE) system, or a zinc fingersystem for inhibiting the expression of PD-1 and, optionally, LAG-3, andfurther wherein a CXCR2 adhesion molecule is inserted by agammaretroviral or lentiviral method into the first population of TILs,second population of TILs, or harvested population of TILs.

According to one embodiment, a method for expanding tumor infiltratinglymphocytes (TILs) into a therapeutic population of TILs comprises:

(a) obtaining a first population of TILs from a tumor resected from apatient by processing a tumor sample obtained from the patient intomultiple tumor fragments;

(b) adding the tumor fragments into a closed system;

(c) performing a first expansion by culturing the first population ofTILs in a cell culture medium comprising IL-2 and optionally comprisinga 4-1BB agonist antibody for about 2 to 5 days;

(d) adding OKT-3, to produce a second population of TILs, wherein thefirst expansion is performed in a closed container providing a firstgas-permeable surface area, wherein the first expansion is performed forabout 1 to 3 days to obtain the second population of TILs, wherein thesecond population of TILs is at least 50-fold greater in number than thefirst population of TILs, and wherein the transition from step (c) tostep (d) occurs without opening the system;

(e) performing a sterile electroporation step on the second populationof TILs, wherein the sterile electroporation step mediates the transferof at least one gene editor;

(f) resting the second population of TILs for about 1 day;

(g) performing a second expansion by supplementing the cell culturemedium of the second population of TILs with additional IL-2, optionallyOKT-3 antibody, optionally an OX40 antibody, and antigen presentingcells (APCs), to produce a third population of TILs, wherein the secondexpansion is performed for about 7 to 11 days to obtain the thirdpopulation of TILs, wherein the second expansion is performed in aclosed container providing a second gas-permeable surface area, andwherein the transition from step (f) to step (g) occurs without openingthe system;

(h) harvesting the therapeutic population of TILs obtained from step (g)to provide a harvested TIL population, wherein the transition from step(g) to step (h) occurs without opening the system, wherein the harvestedpopulation of TILs is a therapeutic population of TILs;

(i) transferring the harvested TIL population to an infusion bag,wherein the transfer from step (h) to (i) occurs without opening thesystem; and

(j) cryopreserving the harvested TIL population using adimethylsulfoxide-based cryopreservation medium,

wherein the electroporation step comprises the delivery of a ClusteredRegularly Interspersed Short Palindromic Repeat (CRISPR) system, aTranscription Activator-Like Effector (TALE) system, or a zinc fingersystem for inhibiting the expression of PD-1 and, optionally, LAG-3, andfurther wherein a CCR4 and/or CCR5 adhesion molecule is inserted by agammaretroviral or lentiviral method into the first population of TILs,second population of TILs, or harvested population of TILs.

According to one embodiment, a method for expanding tumor infiltratinglymphocytes (TILs) into a therapeutic population of TILs comprises:

(a) obtaining a first population of TILs from a tumor resected from apatient by processing a tumor sample obtained from the patient intomultiple tumor fragments;

(b) adding the tumor fragments into a closed system;

(c) performing a first expansion by culturing the first population ofTILs in a cell culture medium comprising IL-2 and optionally comprisinga 4-1BB agonist antibody for about 2 to 5 days;

(d) adding OKT-3, to produce a second population of TILs, wherein thefirst expansion is performed in a closed container providing a firstgas-permeable surface area, wherein the first expansion is performed forabout 1 to 3 days to obtain the second population of TILs, wherein thesecond population of TILs is at least 50-fold greater in number than thefirst population of TILs, and wherein the transition from step (c) tostep (d) occurs without opening the system;

(e) performing a sterile electroporation step on the second populationof TILs, wherein the sterile electroporation step mediates the transferof at least one gene editor;

(f) resting the second population of TILs for about 1 day;

(g) performing a second expansion by supplementing the cell culturemedium of the second population of TILs with additional IL-2, optionallyOKT-3 antibody, optionally an OX40 antibody, and antigen presentingcells (APCs), to produce a third population of TILs, wherein the secondexpansion is performed for about 7 to 11 days to obtain the thirdpopulation of TILs, wherein the second expansion is performed in aclosed container providing a second gas-permeable surface area, andwherein the transition from step (f) to step (g) occurs without openingthe system;

(h) harvesting the therapeutic population of TILs obtained from step (g)to provide a harvested TIL population, wherein the transition from step(g) to step (h) occurs without opening the system, wherein the harvestedpopulation of TILs is a therapeutic population of TILs;

(i) transferring the harvested TIL population to an infusion bag,wherein the transfer from step (h) to (i) occurs without opening thesystem; and

(j) cryopreserving the harvested TIL population using adimethylsulfoxide-based cryopreservation medium,

wherein the electroporation step comprises the delivery of a ClusteredRegularly Interspersed Short Palindromic Repeat (CRISPR) system, aTranscription Activator-Like Effector (TALE) system, or a zinc fingersystem for inhibiting the expression of PD-1 and, optionally, LAG-3, andfurther wherein an adhesion molecule selected from the group consistingof CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof isinserted by a gammaretroviral or lentiviral method into the firstpopulation of TILs, second population of TILs, or harvested populationof TILs.

2. Interleukins

According to additional embodiments, gene-editing methods of the presentinvention may be used to increase the expression of certaininterleukins, such as one or more of IL-2, IL-4, IL-7, IL-10, IL-15, andIL-21. Certain interleukins have been demonstrated to augment effectorfunctions of T cells and mediate tumor control.

According to particular embodiments, expression of one or more of IL-2,IL-4, IL-7, IL-10, IL-15, and IL-21 in TILs is enhanced in accordancewith compositions and methods of the present invention. For example, amethod for expanding tumor infiltrating lymphocytes (TILs) into atherapeutic population of TILs may be carried out in accordance with anyembodiment of the methods described herein (e.g., process 2A or themethods shown in FIGS. 20 and 21), wherein the method comprisesgene-editing at least a portion of the TILs by enhancing the expressionof one or more of IL-2, IL-4, IL-7, IL-10, IL-15, and IL-21. Asdescribed in more detail below, the gene-editing process may comprisethe use of a programmable nuclease that mediates the generation of adouble-strand or single-strand break at an interleukin gene. Forexample, a CRISPR method, a TALE method, or a zinc finger method may beused to enhance the expression of certain interleukins in the TILs.

According to one embodiment, a method for expanding tumor infiltratinglymphocytes (TILs) into a therapeutic population of TILs comprises:

(a) obtaining a first population of TILs from a tumor resected from apatient by processing a tumor sample obtained from the patient intomultiple tumor fragments;

(b) adding the tumor fragments into a closed system;

(c) performing a first expansion by culturing the first population ofTILs in a cell culture medium comprising IL-2 and optionally comprisinga 4-1BB agonist antibody for about 2 to 5 days;

(d) adding OKT-3, to produce a second population of TILs, wherein thefirst expansion is performed in a closed container providing a firstgas-permeable surface area, wherein the first expansion is performed forabout 1 to 3 days to obtain the second population of TILs, wherein thesecond population of TILs is at least 50-fold greater in number than thefirst population of TILs, and wherein the transition from step (c) tostep (d) occurs without opening the system;

(e) performing a sterile electroporation step on the second populationof TILs, wherein the sterile electroporation step mediates the transferof at least one gene editor;

(f) resting the second population of TILs for about 1 day;

(g) performing a second expansion by supplementing the cell culturemedium of the second population of TILs with additional IL-2, optionallyOKT-3 antibody, optionally an OX40 antibody, and antigen presentingcells (APCs), to produce a third population of TILs, wherein the secondexpansion is performed for about 7 to 11 days to obtain the thirdpopulation of TILs, wherein the second expansion is performed in aclosed container providing a second gas-permeable surface area, andwherein the transition from step (f) to step (g) occurs without openingthe system;

(h) harvesting the therapeutic population of TILs obtained from step (g)to provide a harvested TIL population, wherein the transition from step(g) to step (h) occurs without opening the system, wherein the harvestedpopulation of TILs is a therapeutic population of TILs;

(i) transferring the harvested TIL population to an infusion bag,wherein the transfer from step (h) to (i) occurs without opening thesystem; and

(j) cryopreserving the harvested TIL population using adimethylsulfoxide-based cryopreservation medium,

wherein the electroporation step comprises the delivery of a ClusteredRegularly Interspersed Short Palindromic Repeat (CRISPR) system, aTranscription Activator-Like Effector (TALE) system, or a zinc fingersystem for inhibiting the expression of PD-1 and, optionally, LAG-3, andfurther wherein a interleukin selected from the group consisting ofIL-2, IL-4, IL-7, IL-10, IL-15, IL-21, and combinations thereof isinserted by a gammaretroviral or lentiviral method into the firstpopulation of TILs, second population of TILs, or harvested populationof TILs.

E. Gene Editing Methods

As discussed above, embodiments of the present invention provide tumorinfiltrating lymphocytes (TILs) that have been genetically modified viagene-editing to enhance their therapeutic effect. Embodiments of thepresent invention embrace genetic editing through nucleotide insertion(RNA or DNA) into a population of TILs for both promotion of theexpression of one or more proteins and inhibition of the expression ofone or more proteins, as well as combinations thereof. Embodiments ofthe present invention also provide methods for expanding TILs into atherapeutic population, wherein the methods comprise gene-editing theTILs. There are several gene-editing technologies that may be used togenetically modify a population of TILs, which are suitable for use inaccordance with the present invention.

In some embodiments, a method of genetically modifying a population ofTILs includes the step of stable incorporation of genes for productionof one or more proteins. In an embodiment, a method of geneticallymodifying a population of TILs includes the step of retroviraltransduction. In an embodiment, a method of genetically modifying apopulation of TILs includes the step of lentiviral transduction.Lentiviral transduction systems are known in the art and are described,e.g., in Levine, et al., Proc. Nat'l Acad. Sci. 2006, 103, 17372-77;Zufferey, et al., Nat. Biotechnol. 1997, 15, 871-75; Dull, et al., JVirology 1998, 72, 8463-71, and U.S. Pat. No. 6,627,442, the disclosuresof each of which are incorporated by reference herein. In an embodiment,a method of genetically modifying a population of TILs includes the stepof gamma-retroviral transduction. Gamma-retroviral transduction systemsare known in the art and are described, e.g., Cepko and Pear, Cur. Prot.Mol. Biol. 1996, 9.9.1-9.9.16, the disclosure of which is incorporatedby reference herein. In an embodiment, a method of genetically modifyinga population of TILs includes the step of transposon-mediated genetransfer. Transposon-mediated gene transfer systems are known in the artand include systems wherein the transposase is provided as DNAexpression vector or as an expressible RNA or a protein such thatlong-term expression of the transposase does not occur in the transgeniccells, for example, a transposase provided as an mRNA (e.g., an mRNAcomprising a cap and poly-A tail). Suitable transposon-mediated genetransfer systems, including the salmonid-type Tel-like transposase (SBor Sleeping Beauty transposase), such as SB10, SB11, and SB100×, andengineered enzymes with increased enzymatic activity, are described in,e.g., Hackett, et al., Mol. Therapy 2010, 18, 674-83 and U.S. Pat. No.6,489,458, the disclosures of each of which are incorporated byreference herein.

In an embodiment, a method of genetically modifying a population of TILsincludes the step of stable incorporation of genes for production orinhibition (e.g., silencing) of one or more proteins. In an embodiment,a method of genetically modifying a population of TILs includes the stepof electroporation. Electroporation methods are known in the art and aredescribed, e.g., in Tsong, Biophys. J. 1991, 60, 297-306, and U.S.Patent Application Publication No. 2014/0227237 A1, the disclosures ofeach of which are incorporated by reference herein. Otherelectroporation methods known in the art, such as those described inU.S. Pat. Nos. 5,019,034; 5,128,257; 5,137,817; 5,173,158; 5,232,856;5,273,525; 5,304,120; 5,318,514; 6,010,613 and 6,078,490, thedisclosures of which are incorporated by reference herein, may be used.In an embodiment, the electroporation method is a sterileelectroporation method. In an embodiment, the electroporation method isa pulsed electroporation method. In an embodiment, the electroporationmethod is a pulsed electroporation method comprising the steps oftreating TILs with pulsed electrical fields to alter, manipulate, orcause defined and controlled, permanent or temporary changes in theTILs, comprising the step of applying a sequence of at least threesingle, operator-controlled, independently programmed, DC electricalpulses, having field strengths equal to or greater than 100 V/cm, to theTILs, wherein the sequence of at least three DC electrical pulses hasone, two, or three of the following characteristics: (1) at least two ofthe at least three pulses differ from each other in pulse amplitude; (2)at least two of the at least three pulses differ from each other inpulse width; and (3) a first pulse interval for a first set of two ofthe at least three pulses is different from a second pulse interval fora second set of two of the at least three pulses. In an embodiment, theelectroporation method is a pulsed electroporation method comprising thesteps of treating TILs with pulsed electrical fields to alter,manipulate, or cause defined and controlled, permanent or temporarychanges in the TILs, comprising the step of applying a sequence of atleast three single, operator-controlled, independently programmed, DCelectrical pulses, having field strengths equal to or greater than 100V/cm, to the TILs, wherein at least two of the at least three pulsesdiffer from each other in pulse amplitude. In an embodiment, theelectroporation method is a pulsed electroporation method comprising thesteps of treating TILs with pulsed electrical fields to alter,manipulate, or cause defined and controlled, permanent or temporarychanges in the TILs, comprising the step of applying a sequence of atleast three single, operator-controlled, independently programmed, DCelectrical pulses, having field strengths equal to or greater than 100V/cm, to the TILs, wherein at least two of the at least three pulsesdiffer from each other in pulse width. In an embodiment, theelectroporation method is a pulsed electroporation method comprising thesteps of treating TILs with pulsed electrical fields to alter,manipulate, or cause defined and controlled, permanent or temporarychanges in the TILs, comprising the step of applying a sequence of atleast three single, operator-controlled, independently programmed, DCelectrical pulses, having field strengths equal to or greater than 100V/cm, to the TILs, wherein a first pulse interval for a first set of twoof the at least three pulses is different from a second pulse intervalfor a second set of two of the at least three pulses. In an embodiment,the electroporation method is a pulsed electroporation method comprisingthe steps of treating TILs with pulsed electrical fields to induce poreformation in the TILs, comprising the step of applying a sequence of atleast three DC electrical pulses, having field strengths equal to orgreater than 100 V/cm, to TILs, wherein the sequence of at least threeDC electrical pulses has one, two, or three of the followingcharacteristics: (1) at least two of the at least three pulses differfrom each other in pulse amplitude; (2) at least two of the at leastthree pulses differ from each other in pulse width; and (3) a firstpulse interval for a first set of two of the at least three pulses isdifferent from a second pulse interval for a second set of two of the atleast three pulses, such that induced pores are sustained for arelatively long period of time, and such that viability of the TILs ismaintained. In an embodiment, a method of genetically modifying apopulation of TILs includes the step of calcium phosphate transfection.Calcium phosphate transfection methods (calcium phosphate DNAprecipitation, cell surface coating, and endocytosis) are known in theart and are described in Graham and van der Eb, Virology 1973, 52,456-467; Wigler, et al., Proc. Natl. Acad. Sci. 1979, 76, 1373-1376; andChen and Okayarea, Mol. Cell. Biol. 1987, 7, 2745-2752; and in U.S. Pat.No. 5,593,875, the disclosures of each of which are incorporated byreference herein. In an embodiment, a method of genetically modifying apopulation of TILs includes the step of liposomal transfection.Liposomal transfection methods, such as methods that employ a 1:1 (w/w)liposome formulation of the cationic lipidN-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA)and dioleoyl phophotidylethanolamine (DOPE) in filtered water, are knownin the art and are described in Rose, et al., Biotechniques 1991, 10,520-525 and Felgner, et al., Proc. Natl. Acad. Sci. USA, 1987, 84,7413-7417 and in U.S. Pat. Nos. 5,279,833; 5,908,635; 6,056,938;6,110,490; 6,534,484; and 7,687,070, the disclosures of each of whichare incorporated by reference herein. In an embodiment, a method ofgenetically modifying a population of TILs includes the step oftransfection using methods described in U.S. Pat. Nos. 5,766,902;6,025,337; 6,410,517; 6,475,994; and 7,189,705; the disclosures of eachof which are incorporated by reference herein.

According to an embodiment, the gene-editing process may comprise theuse of a programmable nuclease that mediates the generation of adouble-strand or single-strand break at one or more immune checkpointgenes. Such programmable nucleases enable precise genome editing byintroducing breaks at specific genomic loci, i.e., they rely on therecognition of a specific DNA sequence within the genome to target anuclease domain to this location and mediate the generation of adouble-strand break at the target sequence. A double-strand break in theDNA subsequently recruits endogenous repair machinery to the break siteto mediate genome editing by either non-homologous end-joining (NHEJ) orhomology-directed repair (HDR). Thus, the repair of the break can resultin the introduction of insertion/deletion mutations that disrupt (e.g.,silence, repress, or enhance) the target gene product.

Major classes of nucleases that have been developed to enablesite-specific genomic editing include zinc finger nucleases (ZFNs),transcription activator-like nucleases (TALENs), and CRISPR-associatednucleases (e.g., CRISPR/Cas9). These nuclease systems can be broadlyclassified into two categories based on their mode of DNA recognition:ZFNs and TALENs achieve specific DNA binding via protein-DNAinteractions, whereas CRISPR systems, such as Cas9, are targeted tospecific DNA sequences by a short RNA guide molecule that base-pairsdirectly with the target DNA and by protein-DNA interactions. See, e.g.,Cox et al., Nature Medicine, 2015, Vol. 21, No. 2.

Non-limiting examples of gene-editing methods that may be used inaccordance with TIL expansion methods of the present invention includeCRISPR methods, TALE methods, and ZFN methods, embodiments of which aredescribed in more detail below. According to an embodiment, a method forexpanding TILs into a therapeutic population may be carried out inaccordance with any embodiment of the methods described herein (e.g.,process 2A) or as described in PCT/US2017/058610, PCT/US2018/012605, orPCT/US2018/012633, wherein the method further comprises gene-editing atleast a portion of the TILs by one or more of a CRISPR method, a TALEmethod or a ZFN method, in order to generate TILs that can provide anenhanced therapeutic effect. According to an embodiment, gene-editedTILs can be evaluated for an improved therapeutic effect by comparingthem to non-modified TILs in vitro, e.g., by evaluating in vitroeffector function, cytokine profiles, etc. compared to unmodified TILs.

In some embodiments of the present invention, electroporation is usedfor delivery of a gene editing system, such as CRISPR, TALEN, and ZFNsystems. In some embodiments of the present invention, theelectroporation system is a flow electroporation system. An example of asuitable flow electroporation system suitable for use with someembodiments of the present invention is the commercially-availableMaxCyte STX system. There are several alternative commercially-availableelectroporation instruments which may be suitable for use with thepresent invention, such as the AgilePulse system or ECM 830 availablefrom BTX-Harvard Apparatus, Cellaxess Elektra (Cellectricon),Nucleofector (Lonza/Amaxa), GenePulser MXcell (BIORAD), iPorator-96(Primax) or siPORTer96 (Ambion). In some embodiments of the presentinvention, the electroporation system forms a closed, sterile systemwith the remainder of the TIL expansion method. In some embodiments ofthe present invention, the electroporation system is a pulsedelectroporation system as described herein, and forms a closed, sterilesystem with the remainder of the TIL expansion method.

1. CRISPR Methods

A method for expanding TILs into a therapeutic population may be carriedout in accordance with any embodiment of the methods described herein(e.g., process 2A) or as described in PCT/US2017/058610,PCT/US2018/012605, or PCT/US2018/012633, wherein the method furthercomprises gene-editing at least a portion of the TILs by a CRISPR method(e.g., CRISPR/Cas9 or CRISPR/Cpf1). According to particular embodiments,the use of a CRISPR method during the TIL expansion process causesexpression of one or more immune checkpoint genes to be silenced orreduced in at least a portion of the therapeutic population of TILs.Alternatively, the use of a CRISPR method during the TIL expansionprocess causes expression of one or more immune checkpoint genes to beenhanced in at least a portion of the therapeutic population of TILs.

CRISPR stands for “Clustered Regularly Interspaced Short PalindromicRepeats.” A method of using a CRISPR system for gene editing is alsoreferred to herein as a CRISPR method. CRISPR systems can be dividedinto two main classes, Class 1 and Class 2, which are further classifiedinto different types and sub-types. The classification of the CRISPRsystems is based on the effector Cas proteins that are capable ofcleaving specific nucleic acids. In Class 1 CRISPR systems the effectormodule consists of a multi-protein complex, whereas Class 2 systems onlyuse one effector protein. Class 1 CRISPR includes Types I, III, and IVand Class 2 CRISPR includes Types II, V, and VI. While any of thesetypes of CRISPR systems may be used in accordance with the presentinvention, there are three types of CRISPR systems which incorporateRNAs and Cas proteins that are preferred for use in accordance with thepresent invention: Types I (exemplified by Cas3), II (exemplified byCas9), and III (exemplified by Cas10). The Type II CRISPR is one of themost well-characterized systems.

CRISPR technology was adapted from the natural defense mechanisms ofbacteria and archaea (the domain of single-celled microorganisms). Theseorganisms use CRISPR-derived RNA and various Cas proteins, includingCas9, to foil attacks by viruses and other foreign bodies by chopping upand destroying the DNA of a foreign invader. A CRISPR is a specializedregion of DNA with two distinct characteristics: the presence ofnucleotide repeats and spacers. Repeated sequences of nucleotides aredistributed throughout a CRISPR region with short segments of foreignDNA (spacers) interspersed among the repeated sequences. In the type IICRISPR/Cas system, spacers are integrated within the CRISPR genomic lociand transcribed and processed into short CRISPR RNA (crRNA). ThesecrRNAs anneal to trans-activating crRNAs (tracrRNAs) and directsequence-specific cleavage and silencing of pathogenic DNA by Casproteins. Target recognition by the Cas9 protein requires a “seed”sequence within the crRNA and a conserved dinucleotide-containingprotospacer adjacent motif (PAM) sequence upstream of the crRNA-bindingregion. The CRISPR/Cas system can thereby be retargeted to cleavevirtually any DNA sequence by redesigning the crRNA. Thus, according tocertain embodiments, Cas9 serves as an RNA-guided DNA endonuclease thatcleaves DNA upon crRNA-tracrRNA recognition.

The crRNA and tracrRNA in the native system can be simplified into asingle guide RNA (sgRNA) of approximately 100 nucleotides for use ingenetic engineering. The sgRNA is a synthetic RNA that includes ascaffold sequence necessary for Cas-binding and a user-definedapproximately 17- to 20-nucleotide spacer that defines the genomictarget to be modified. Thus, a user can change the genomic target of theCas protein by changing the target sequence present in the sgRNA. TheCRISPR/Cas system is directly portable to human cells by co-delivery ofplasmids expressing the Cas9 endo-nuclease and the RNA components (e.g.,sgRNA). Different variants of Cas proteins may be used to reducetargeting limitations (e.g., orthologs of Cas9, such as Cpf1).

According to one embodiment, an engineered, programmable, non-naturallyoccurring Type II CRISPR-Cas system comprises a Cas9 protein and atleast one guide RNA that targets and hybridizes to a target sequence ofa DNA molecule in a TIL, wherein the DNA molecule encodes and the TILexpresses at least one immune checkpoint molecule and the Cas9 proteincleaves the DNA molecules, whereby expression of the at least one immunecheckpoint molecule is altered; and, wherein the Cas9 protein and theguide RNA do not naturally occur together. According to an embodiment,the expression of two or more immune checkpoint molecules is altered.According to an embodiment, the guide RNA(s) comprise a guide sequencefused to a tracr sequence. For example, the guide RNA may comprisecrRNA-tracrRNA or sgRNA. According to aspects of the present invention,the terms “guide RNA”, “single guide RNA” and “synthetic guide RNA” maybe used interchangeably and refer to the polynucleotide sequencecomprising the guide sequence, which is the approximately 17-20 bpsequence within the guide RNA that specifies the target site.

Variants of Cas9 having improved on-target specificity compared to Cas9may also be used in accordance with embodiments of the presentinvention. Such variants may be referred to as high-fidelity Cas-9s.According to an embodiment, a dual nickase approach may be utilized,wherein two nickases targeting opposite DNA strands generate a DSBwithin the target DNA (often referred to as a double nick or dualnickase CRISPR system). For example, this approach may involve themutation of one of the two Cas9 nuclease domains, turning Cas9 from anuclease into a nickase. Non-limiting examples of high-fidelity Cas9sinclude eSpCas9, SpCas9-HF1 and HypaCas9. Such variants may reduce oreliminate unwanted changes at non-target DNA sites. See, e.g., SlaymakerI M, et al. Science. 2015 Dec. 1, Kleinstiver B P, et al. Nature. 2016Jan. 6, and Ran et al., Nat Protoc. 2013 November; 8(11):2281-2308, thedisclosures of which are incorporated by reference herein.

Additionally, according to particular embodiments, Cas9 scaffolds may beused that improve gene delivery of Cas9 into cells and improve on-targetspecificity, such as those disclosed in U.S. Patent ApplicationPublication No. 2016/0102324, which is incorporated by reference herein.For example, Cas9 scaffolds may include a RuvC motif as defined by(D-[I/L]-G-X-X-S-X-G-W-A) and/or a HNH motif defined by(Y-X-X-D-H-X-X-P-X-S-X-X-X-D-X-S), where X represents any one of the 20naturally occurring amino acids and [I/L] represents isoleucine orleucine. The HNH domain is responsible for nicking one strand of thetarget dsDNA and the RuvC domain is involved in cleavage of the otherstrand of the dsDNA. Thus, each of these domains nick a strand of thetarget DNA within the protospacer in the immediate vicinity of PAM,resulting in blunt cleavage of the DNA. These motifs may be combinedwith each other to create more compact and/or more specific Cas9scaffolds. Further, the motifs may be used to create a split Cas9protein (i.e., a reduced or truncated form of a Cas9 protein or Cas9variant that comprises either a RuvC domain or a HNH domain) that isdivided into two separate RuvC and HNH domains, which can process thetarget DNA together or separately.

According to particular embodiments, a CRISPR method comprises silencingor reducing the expression of one or more immune checkpoint genes inTILs by introducing a Cas9 nuclease and a guide RNA (e.g.,crRNA-tracrRNA or sgRNA) containing a sequence of approximately 17-20nucleotides specific to a target DNA sequence of the immune checkpointgene(s). The guide RNA may be delivered as RNA or by transforming aplasmid with the guide RNA-coding sequence under a promoter. TheCRISPR/Cas enzymes introduce a double-strand break (DSB) at a specificlocation based on a sgRNA-defined target sequence. DSBs may be repairedin the cells by non-homologous end joining (NHEJ), a mechanism whichfrequently causes insertions or deletions (indels) in the DNA. Indelsoften lead to frameshifts, creating loss of function alleles; forexample, by causing premature stop codons within the open reading frame(ORF) of the targeted gene. According to certain embodiments, the resultis a loss-of-function mutation within the targeted immune checkpointgene.

Alternatively, DSBs induced by CRISPR/Cas enzymes may be repaired byhomology-directed repair (HDR) instead of NHEJ. While NHEJ-mediated DSBrepair often disrupts the open reading frame of the gene, homologydirected repair (HDR) can be used to generate specific nucleotidechanges ranging from a single nucleotide change to large insertions.According to an embodiment, HDR is used for gene editing immunecheckpoint genes by delivering a DNA repair template containing thedesired sequence into the TILs with the sgRNA(s) and Cas9 or Cas9nickase. The repair template preferably contains the desired edit aswell as additional homologous sequence immediately upstream anddownstream of the target gene (often referred to as left and righthomology arms).

According to particular embodiments, an enzymatically inactive versionof Cas9 (deadCas9 or dCas9) may be targeted to transcription start sitesin order to repress transcription by blocking initiation. Thus, targetedimmune checkpoint genes may be repressed without the use of a DSB. AdCas9 molecule retains the ability to bind to target DNA based on thesgRNA targeting sequence. According to an embodiment of the presentinvention, a CRISPR method comprises silencing or reducing theexpression of one or more immune checkpoint genes by inhibiting orpreventing transcription of the targeted gene(s). For example, a CRISPRmethod may comprise fusing a transcriptional repressor domain, such as aKruppel-associated box (KRAB) domain, to an enzymatically inactiveversion of Cas9, thereby forming, e.g., a dCas9-KRAB, that targets theimmune checkpoint gene's transcription start site, leading to theinhibition or prevention of transcription of the gene. Preferably, therepressor domain is targeted to a window downstream from thetranscription start site, e.g., about 500 bp downstream. This approach,which may be referred to as CRISPR interference (CRISPRi), leads torobust gene knockdown via transcriptional reduction of the target RNA.

According to particular embodiments, an enzymatically inactive versionof Cas9 (deadCas9 or dCas9) may be targeted to transcription start sitesin order to activate transcription. This approach may be referred to asCRISPR activation (CRISPRa). According to an embodiment, a CRISPR methodcomprises increasing the expression of one or more immune checkpointgenes by activating transcription of the targeted gene(s). According tosuch embodiments, targeted immune checkpoint genes may be activatedwithout the use of a DSB. A CRISPR method may comprise targetingtranscriptional activation domains to the transcription start site; forexample, by fusing a transcriptional activator, such as VP64, to dCas9,thereby forming, e.g., a dCas9-VP64, that targets the immune checkpointgene's transcription start site, leading to activation of transcriptionof the gene. Preferably, the activator domain is targeted to a windowupstream from the transcription start site, e.g., about 50-400 bpdownstream

Additional embodiments of the present invention may utilize activationstrategies that have been developed for potent activation of targetgenes in mammalian cells. Non-limiting examples include co-expression ofepitope-tagged dCas9 and antibody-activator effector proteins (e.g., theSunTag system), dCas9 fused to a plurality of different activationdomains in series (e.g., dCas9-VPR) or co-expression of dCas9-VP64 witha modified scaffold gRNA and additional RNA-binding helper activators(e.g., SAM activators).

According to other embodiments, a CRISPR-mediated genome editing methodreferred to as CRISPR assisted rational protein engineering (CARPE) maybe used in accordance with embodiments of the present invention, asdisclosed in U.S. Pat. No. 9,982,278, which is incorporated by referenceherein. CARPE involves the generation of “donor” and “destination”libraries that incorporate directed mutations from single-stranded DNA(ssDNA) or double-stranded DNA (dsDNA) editing cassettes directly intothe genome. Construction of the donor library involves cotransformingrationally designed editing oligonucleotides into cells with a guide RNA(gRNA) that hybridizes to a target DNA sequence. The editingoligonucleotides are designed to couple deletion or mutation of a PAMwith the mutation of one or more desired codons in the adjacent gene.This enables the entire donor library to be generated in a singletransformation. The donor library is retrieved by amplification of therecombinant chromosomes, such as by a PCR reaction, using a syntheticfeature from the editing oligonucleotide, namely, a second PAM deletionor mutation that is simultaneously incorporated at the 3′ terminus ofthe gene. This covalently couples the codon target mutations directed toa PAM deletion. The donor libraries are then co-transformed into cellswith a destination gRNA vector to create a population of cells thatexpress a rationally designed protein library.

According to other embodiments, methods for trackable, precision genomeediting using a CRISPR-mediated system referred to as Genome Engineeringby Trackable CRISPR Enriched Recombineering (GEn-TraCER) may be used inaccordance with embodiments of the present invention, as disclosed inU.S. Pat. No. 9,982,278, which is incorporated by reference herein. TheGEn-TraCER methods and vectors combine an editing cassette with a geneencoding gRNA on a single vector. The cassette contains a desiredmutation and a PAM mutation. The vector, which may also encode Cas9, isthe introduced into a cell or population of cells. This activatesexpression of the CRISPR system in the cell or population of cells,causing the gRNA to recruit Cas9 to the target region, where a dsDNAbreak occurs, allowing integration of the PAM mutation.

Non-limiting examples of genes that may be silenced or inhibited bypermanently gene-editing TILs via a CRISPR method include PD-1, CTLA-4,LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6,PTPN22, PDCD1, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9,CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD,FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB,HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF,GUCY1A2, GUCY1A3, GUCY1B2, and GUCY1B3.

Non-limiting examples of genes that may be enhanced by permanentlygene-editing TILs via a CRISPR method include CCR2, CCR4, CCR5, CXCR2,CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-21, the NOTCH 1/2intracellular domain (ICD), and/or the NOTCH ligand mDLL1.

Examples of systems, methods, and compositions for altering theexpression of a target gene sequence by a CRISPR method, and which maybe used in accordance with embodiments of the present invention, aredescribed in U.S. Pat. Nos. 8,697,359; 8,993,233; 8,795,965; 8,771,945;8,889,356; 8,865,406; 8,999,641; 8,945,839; 8,932,814; 8,871,445;8,906,616; and 8,895,308, which are incorporated by reference herein.Resources for carrying out CRISPR methods, such as plasmids forexpressing CRISPR/Cas9 and CRISPR/Cpf1, are commercially available fromcompanies such as GenScript.

In an embodiment, genetic modifications of populations of TILs, asdescribed herein, may be performed using the CRISPR/Cpf1 system asdescribed in U.S. Pat. No. 9,790,490, the disclosure of which isincorporated by reference herein. The CRISPR/Cpf1 system is functionallydistinct from the CRISPR-Cas9 system in that Cpf1-associated CRISPRarrays are processed into mature crRNAs without the need for anadditional tracrRNA. The crRNAs used in the CRISPR/Cpf1 system have aspacer or guide sequence and a direct repeat sequence. The Cpf1p-crRNAcomplex that is formed using this method is sufficient by itself tocleave the target DNA.

According to one embodiment, a method for expanding tumor infiltratinglymphocytes (TILs) into a therapeutic population of TILs comprises:

(a) obtaining a first population of TILs from a tumor resected from apatient by processing a tumor sample obtained from the patient intomultiple tumor fragments;

(b) adding the tumor fragments into a closed system;

(c) performing a first expansion by culturing the first population ofTILs in a cell culture medium comprising IL-2 and optionally comprisinga 4-1BB agonist antibody for about 2 to 5 days;

(d) adding OKT-3, to produce a second population of TILs, wherein thefirst expansion is performed in a closed container providing a firstgas-permeable surface area, wherein the first expansion is performed forabout 1 to 3 days to obtain the second population of TILs, wherein thesecond population of TILs is at least 50-fold greater in number than thefirst population of TILs, and wherein the transition from step (c) tostep (d) occurs without opening the system;

(e) performing a sterile electroporation step on the second populationof TILs, wherein the sterile electroporation step mediates the transferof at least one gene editor;

(f) resting the second population of TILs for about 1 day;

(g) performing a second expansion by supplementing the cell culturemedium of the second population of TILs with additional IL-2, optionallyOKT-3 antibody, optionally an OX40 antibody, and antigen presentingcells (APCs), to produce a third population of TILs, wherein the secondexpansion is performed for about 7 to 11 days to obtain the thirdpopulation of TILs, wherein the second expansion is performed in aclosed container providing a second gas-permeable surface area, andwherein the transition from step (f) to step (g) occurs without openingthe system;

(h) harvesting the therapeutic population of TILs obtained from step (g)to provide a harvested TIL population, wherein the transition from step(g) to step (h) occurs without opening the system, wherein the harvestedpopulation of TILs is a therapeutic population of TILs;

(i) transferring the harvested TIL population to an infusion bag,wherein the transfer from step (h) to (i) occurs without opening thesystem; and

(j) cryopreserving the harvested TIL population using adimethylsulfoxide-based cryopreservation medium,

wherein the electroporation step comprises the delivery of a ClusteredRegularly Interspersed Short Palindromic Repeat (CRISPR)/Cas9 orCRISPR/Cpf1 system for modulating the expression of at least oneprotein.

According to one embodiment, a method for expanding tumor infiltratinglymphocytes (TILs) into a therapeutic population of TILs comprises:

(a) obtaining a first population of TILs from a tumor resected from apatient by processing a tumor sample obtained from the patient intomultiple tumor fragments;

(b) adding the tumor fragments into a closed system;

(c) performing a first expansion by culturing the first population ofTILs in a cell culture medium comprising IL-2 and optionally comprisinga 4-1BB agonist antibody for about 2 to 5 days;

(d) adding OKT-3, to produce a second population of TILs, wherein thefirst expansion is performed in a closed container providing a firstgas-permeable surface area, wherein the first expansion is performed forabout 1 to 3 days to obtain the second population of TILs, wherein thesecond population of TILs is at least 50-fold greater in number than thefirst population of TILs, and wherein the transition from step (c) tostep (d) occurs without opening the system;

(e) performing a sterile electroporation step on the second populationof TILs, wherein the sterile electroporation step mediates the transferof at least one gene editor;

(f) resting the second population of TILs for about 1 day;

(g) performing a second expansion by supplementing the cell culturemedium of the second population of TILs with additional IL-2, optionallyOKT-3 antibody, optionally an OX40 antibody, and antigen presentingcells (APCs), to produce a third population of TILs, wherein the secondexpansion is performed for about 7 to 11 days to obtain the thirdpopulation of TILs, wherein the second expansion is performed in aclosed container providing a second gas-permeable surface area, andwherein the transition from step (f) to step (g) occurs without openingthe system;

(h) harvesting the therapeutic population of TILs obtained from step (g)to provide a harvested TIL population, wherein the transition from step(g) to step (h) occurs without opening the system, wherein the harvestedpopulation of TILs is a therapeutic population of TILs;

(i) transferring the harvested TIL population to an infusion bag,wherein the transfer from step (h) to (i) occurs without opening thesystem; and

(j) cryopreserving the harvested TIL population using adimethylsulfoxide-based cryopreservation medium,

wherein the electroporation step comprises the delivery of a ClusteredRegularly Interspersed Short Palindromic Repeat (CRISPR)/Cas9 orCRISPR/Cpf1 system for inhibiting the expression of PD-1 and LAG-3.

2. TALE Methods

A method for expanding TILs into a therapeutic population may be carriedout in accordance with any embodiment of the methods described herein(e.g., process 2A) or as described in PCT/US2017/058610,PCT/US2018/012605, or PCT/US2018/012633, wherein the method furthercomprises gene-editing at least a portion of the TILs by a TALE method.According to particular embodiments, the use of a TALE method during theTIL expansion process causes expression of one or more immune checkpointgenes to be silenced or reduced in at least a portion of the therapeuticpopulation of TILs. Alternatively, the use of a TALE method during theTIL expansion process causes expression of one or more immune checkpointgenes to be enhanced in at least a portion of the therapeutic populationof TILs.

TALE stands for “Transcription Activator-Like Effector” proteins, whichinclude TALENs (“Transcription Activator-Like Effector Nucleases”). Amethod of using a TALE system for gene editing may also be referred toherein as a TALE method. TALEs are naturally occurring proteins from theplant pathogenic bacteria genus Xanthomonas, and contain DNA-bindingdomains composed of a series of 33-35-amino-acid repeat domains thateach recognizes a single base pair. TALE specificity is determined bytwo hypervariable amino acids that are known as the repeat-variabledi-residues (RVDs). Modular TALE repeats are linked together torecognize contiguous DNA sequences. A specific RVD in the DNA-bindingdomain recognizes a base in the target locus, providing a structuralfeature to assemble predictable DNA-binding domains. The DNA bindingdomains of a TALE are fused to the catalytic domain of a type IIS FokIendonuclease to make a targetable TALE nuclease. To induce site-specificmutation, two individual TALEN arms, separated by a 14-20 base pairspacer region, bring FokI monomers in close proximity to dimerize andproduce a targeted double-strand break.

Several large, systematic studies utilizing various assembly methodshave indicated that TALE repeats can be combined to recognize virtuallyany user-defined sequence. Strategies that enable the rapid assembly ofcustom TALE arrays include Golden Gate molecular cloning,high-throughput solid-phase assembly, and ligation-independent cloningtechniques. Custom-designed TALE arrays are also commercially availablethrough Cellectis Bioresearch (Paris, France), TransposagenBiopharmaceuticals (Lexington, Ky., USA), and Life Technologies (GrandIsland, N.Y., USA). Additionally web-based tools, such as TALEffector-Nucleotide Target 2.0, are available that enable the design ofcustom TAL effector repeat arrays for desired targets and also providespredicted TAL effector binding sites. See Doyle, et al., Nucleic AcidsResearch, 2012, Vol. 40, W117-W122. Examples of TALE and TALEN methodssuitable for use in the present invention are described in U.S. PatentApplication Publication Nos. US 2011/0201118 A1; US 2013/0117869 A1; US2013/0315884 A1; US 2015/0203871 A1 and US 2016/0120906 A1, thedisclosures of which are incorporated by reference herein.

According to an embodiment of the present invention, a TALE methodcomprises silencing or reducing the expression of one or more immunecheckpoint genes by inhibiting or preventing transcription of thetargeted gene(s). For example, a TALE method may include utilizingKRAB-TALEs, wherein the method comprises fusing a transcriptionalKruppel-associated box (KRAB) domain to a DNA binding domain thattargets the gene's transcription start site, leading to the inhibitionor prevention of transcription of the gene.

According to another embodiment, a TALE method comprises silencing orreducing the expression of one or more immune checkpoint genes byintroducing mutations in the targeted gene(s). For example, a TALEmethod may include fusing a nuclease effector domain, such as Fokl, tothe TALE DNA binding domain, resulting in a TALEN. Fokl is active as adimer; hence, the method comprises constructing pairs of TALENs toposition the FOKL nuclease domains to adjacent genomic target sites,where they introduce DNA double strand breaks. A double strand break maybe completed following correct positioning and dimerization of Fokl.Once the double strand break is introduced, DNA repair can be achievedvia two different mechanisms: the high-fidelity homologous recombinationpair (HRR) (also known as homology-directed repair or HDR) or theerror-prone non-homologous end joining (NHEJ). Repair of double strandbreaks via NHEJ preferably results in DNA target site deletions,insertions or substitutions, i.e., NHEJ typically leads to theintroduction of small insertions and deletions at the site of the break,often inducing frameshifts that knockout gene function. According toparticular embodiments, the TALEN pairs are targeted to the most 5′exons of the genes, promoting early frame shift mutations or prematurestop codons. The genetic mutation(s) introduced by TALEN are preferablypermanent. Thus, according to one embodiment, the method comprisessilencing or reducing expression of an immune checkpoint gene byutilizing dimerized TALENs to induce a site-specific double strand breakthat is repaired via error-prone NHEJ, leading to one or more mutationsin the targeted immune checkpoint gene.

According to additional embodiments, TALENs are utilized to introducegenetic alterations via HRR, such as non-random point mutations,targeted deletion, or addition of DNA fragments. The introduction of DNAdouble strand breaks enables gene editing via homologous recombinationin the presence of suitable donor DNA. According to an embodiment, themethod comprises co-delivering dimerized TALENs and a donor plasmidbearing locus-specific homology arms to induce a site-specific doublestrand break and integrate one or more transgenes into the DNA.

According to another embodiment, a TALEN that is a hybrid proteinderived from FokI and AvrXa7, as disclosed in U.S. Patent PublicationNo. 2011/0201118, may be used in accordance with embodiments of thepresent invention. This TALEN retains recognition specificity for targetnucleotides of AvrXa7 and the double-stranded DNA cleaving activity ofFokI. The same methods can be used to prepare other TALEN havingdifferent recognition specificity. For example, compact TALENs may begenerated by engineering a core TALE scaffold having different sets ofRVDs to change the DNA binding specificity and target a specific singledsDNA target sequence. See U.S. Patent Publication No. 2013/0117869. Aselection of catalytic domains can be attached to the scaffold to effectDNA processing, which may be engineered to ensure that the catalyticdomain is capable of processing DNA near the single dsDNA targetsequence when fused to the core TALE scaffold. A peptide linker may alsobe engineered to fuse the catalytic domain to the scaffold to create acompact TALEN made of a single polypeptide chain that does not requiredimerization to target a specific single dsDNA sequence. A core TALEscaffold may also be modified by fusing a catalytic domain, which may bea TAL monomer, to its N-terminus, allowing for the possibility that thiscatalytic domain might interact with another catalytic domain fused toanother TAL monomer, thereby creating a catalytic entity likely toprocess DNA in the proximity of the target sequences. See U.S. PatentPublication No. 2015/0203871. This architecture allows only one DNAstrand to be targeted, which is not an option for classical TALENarchitectures.

According to an embodiment of the present invention, conventional RVDsmay be used create TALENs that are capable of significantly reducinggene expression. In an embodiment, four RVDs, NI, HD, NN, and NG, areused to target adenine, cytosine, guanine, and thymine, respectively.These conventional RVDs can be used to, for instance, create TALENstargeting the PD-1 gene. Examples of TALENs using conventional RVDsinclude the T3v1 and T1 TALENs disclosed in Gautron et al., MolecularTherapy: Nucleic Acids December 2017, Vol. 9:312-321 (Gautron), which isincorporated by reference herein. The T3v1 and T1 TALENs target thesecond exon of the PDCD1 locus where the PD-L1 binding site is locatedand are able to considerably reduce PD-1 production. In an embodiment,the T1 TALEN does so by using target SEQ ID NO:127 and the T3v1 TALENdoes so by using target SEQ ID NO:128.

According to another embodiment, TALENs are modified usingnon-conventional RVDs to improve their activity and specificity for atarget gene, such as disclosed in Gautron. Naturally occurring RVDs onlycover a small fraction of the potential diversity repertoire for thehypervariable amino acid locations. Non-conventional RVDs provide analternative to natural RVDs and have novel intrinsic targetingspecificity features that can be used to exclude the targeting ofoff-site targets (sequences within the genome that contain a fewmismatches relative to the targeted sequence) by TALEN. Non-conventionalRVDs may be identified by generating and screening collections of TALENcontaining alternative combinations of amino acids at the twohypervariable amino acid locations at defined positions of an array asdisclosed in Juillerat, et al., Scientific Reports 5, Article Number8150 (2015), which is incorporated by reference herein. Next,non-conventional RVDs may be selected that discriminate between thenucleotides present at the position of mismatches, which can preventTALEN activity at off-site sequences while still allowing appropriateprocessing of the target location. The selected non-conventional RVDsmay then be used to replace the conventional RVDs in a TALEN. Examplesof TALENs where conventional RVDs have been replaced by non-conventionalRVDs include the T3v2 and T3v3 PD-1 TALENs produced by Gautron. TheseTALENs had increased specificity when compared to TALENs usingconventional RVDs.

According to additional embodiments, TALEN may be utilized to introducegenetic alterations to silence or reduce the expression of two genes.For instance, two separate TALEN may be generated to target twodifferent genes and then used together. The molecular events generatedby the two TALEN at their respective loci and potential off-target sitesmay be characterized by high-throughput DNA sequencing. This enables theanalysis of off-target sites and identification of the sites that mightresult from the use of both TALEN. Based on this information,appropriate conventional and non-conventional RVDs may be selected toengineer TALEN that have increased specificity and activity even whenused together. For example, Gautron discloses the combined use of T3v4PD-1 and TRAC TALEN to produce double knockout CAR T cells, whichmaintained a potent in vitro anti-tumor function.

In an embodiment, the method of Gautron or other methods describedherein may be employed to genetically-edit TILs, which may then beexpanded by any of the procedures described herein. In an embodiment, amethod for expanding tumor infiltrating lymphocytes (TILs) into atherapeutic population of TILs comprises the steps of:

-   -   (a) activating a first population of TILs obtained from a tumor        resected from a patient using CD3 and CD28 activating beads or        antibodies for 1 to 5 days;    -   (b) gene-editing at least a portion of the first population of        TILs using electroporation of transcription activator-like        effector nucleases to obtain a second population of TILs;    -   (c) optionally incubating the second population of TILs;    -   (d) performing a first expansion by culturing the second        population of TILs in a cell culture medium comprising IL-2, and        optionally OKT-3, to produce a third population of TILs, wherein        the first expansion is performed in a closed container providing        a first gas-permeable surface area, wherein the first expansion        is performed for about 3 to 14 days to obtain the third        population of TILs;    -   (e) performing a second expansion by supplementing the cell        culture medium of the third population of TILs with additional        IL-2, OKT-3, and antigen presenting cells (APCs), to produce a        fourth population of TILs, wherein the second expansion is        performed for about 7 to 14 days to obtain the fourth population        of TILs, wherein the fourth population of TILs is a therapeutic        population of TILs;    -   (f) harvesting the therapeutic population of TILs obtained from        step (e);    -   (g) transferring the harvested TIL population from step (e) to        an infusion bag, wherein the transfer from step (e) to (f)        occurs without opening the system; and    -   (h) wherein one or more of steps (a) to (g) are performed in a        closed, sterile system.

In an embodiment, a method for expanding tumor infiltrating lymphocytes(TILs) into a therapeutic population of TILs comprises the steps of:

-   -   (a) activating a first population of TILs obtained from a tumor        resected from a patient using CD3 and CD28 activating beads or        antibodies for 1 to 5 days;    -   (b) gene-editing at least a portion of the first population of        TILs using electroporation of transcription activator-like        effector nucleases in cytoporation medium to obtain a second        population of TILs;    -   (c) optionally incubating the second population of TILs;    -   (d) performing a first expansion by culturing the second        population of TILs in a cell culture medium comprising IL-2, and        optionally OKT-3, to produce a third population of TILs, wherein        the first expansion is performed in a closed container providing        a first gas-permeable surface area, wherein the first expansion        is performed for about 6 to 9 days to obtain the third        population of TILs;    -   (e) performing a second expansion by supplementing the cell        culture medium of the third population of TILs with additional        IL-2, OKT-3, and antigen presenting cells (APCs), to produce a        fourth population of TILs, wherein the second expansion is        performed for about 9 to 11 days to obtain the fourth population        of TILs, wherein the fourth population of TILs is a therapeutic        population of TILs;    -   (f) harvesting the therapeutic population of TILs obtained from        step (e);    -   (g) transferring the harvested TIL population from step (e) to        an infusion bag, wherein the transfer from step (e) to (f)        occurs without opening the system; and    -   (h) wherein one or more of steps (a) to (g) are performed in a        closed, sterile system.

In an embodiment, a method for expanding tumor infiltrating lymphocytes(TILs) into a therapeutic population of TILs comprises the steps of:

-   -   (a) activating a first population of TILs obtained from a tumor        resected from a patient using CD3 and CD28 activating beads or        antibodies for 1 to 5 days;    -   (b) gene-editing at least a portion of the first population of        TILs using electroporation of transcription activator-like        effector nucleases in cytoporation medium to obtain a second        population of TILs;    -   (c) optionally incubating the second population of TILs, wherein        the incubation is performed at about 30-40° C. with about 5%        CO₂;    -   (d) performing a first expansion by culturing the second        population of TILs in a cell culture medium comprising IL-2, and        optionally OKT-3, to produce a third population of TILs, wherein        the first expansion is performed in a closed container providing        a first gas-permeable surface area, wherein the first expansion        is performed for about 6 to 9 days to obtain the third        population of TILs;    -   (e) performing a second expansion by supplementing the cell        culture medium of the third population of TILs with additional        IL-2, OKT-3, and antigen presenting cells (APCs), to produce a        fourth population of TILs, wherein the second expansion is        performed for about 9 to 11 days to obtain the fourth population        of TILs, wherein the fourth population of TILs is a therapeutic        population of TILs;    -   (f) harvesting the therapeutic population of TILs obtained from        step (e);    -   (g) transferring the harvested TIL population from step (e) to        an infusion bag, wherein the transfer from step (e) to (f)        occurs without opening the system; and    -   (h) wherein one or more of steps (a) to (g) are performed in a        closed, sterile system.

According to another embodiment, TALENs may be specifically designed,which allows higher rates of DSB events within the target cell(s) thatare able to target a specific selection of genes. See U.S. PatentPublication No. 2013/0315884. The use of such rare cutting endonucleasesincreases the chances of obtaining double inactivation of target genesin transfected cells, allowing for the production of engineered cells,such as T-cells. Further, additional catalytic domains can be introducedwith the TALEN to increase mutagenesis and enhance target geneinactivation. The TALENs described in U.S. Patent Publication No.2013/0315884 were successfully used to engineer T-cells to make themsuitable for immunotherapy. TALENs may also be used to inactivatevarious immune checkpoint genes in T-cells, including the inactivationof at least two genes in a single T-cell. See U.S. Patent PublicationNo. 2016/0120906. Additionally, TALENs may be used to inactivate genesencoding targets for immunosuppressive agents and T-cell receptors, asdisclosed in U.S. Patent Publication No. 2018/0021379, which isincorporated by reference herein. Further, TALENs may be used to inhibitthe expression of beta 2-microglobulin (B2M) and/or class II majorhistocompatibility complex transactivator (CIITA), as disclosed in U.S.Patent Publication No. 2019/0010514, which is incorporated by referenceherein.

Non-limiting examples of genes that may be silenced or inhibited bypermanently gene-editing TILs via a TALE method include PD-1, CTLA-4,LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6,PTPN22, PDCD1, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9,CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD,FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB,HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF,GUCY1A2, GUCY1A3, GUCY1B2, and GUCY1B3.

Non-limiting examples of TALE-nucleases targeting the PD-1 gene areprovided in the following table. In these examples, the targeted genomicsequences contain two 17-base pair (bp) long sequences (referred to ashalf targets, shown in upper case letters) separated by a 15-bp spacer(shown in lower case letters). Each half target is recognized by repeatsof half TALE-nucleases listed in the table. Thus, according toparticular embodiments, TALE-nucleases according to the inventionrecognize and cleave the target sequence selected from the groupconsisting of: SEQ ID NO: 127 and SEQ ID NO: 128. TALEN sequences andgene-editing methods are also described in Gautron, discussed above.

No. Target PD-1 Sequence Repeat Sequence Half-TALE nuclease 1TTCTCCCCAGCCCTGCTcgtggtgacc Repeat PD-1-left PD-1-left TALENgaagGGACAACGCCACCTTCA (SEQ ID NO: 129) (SEQ ID NO: 133) (SEQ ID NO: 127)Repeat PD-1-right PD-1-right TALEN (SEQ ID NO: 130) (SEQ ID NO: 134) 2TACCTCTGTGGGGCCATctccctggcc Repeat PD-1-left PD-1-left TALENcccaaGGCGCAGATCAAAGAGA (SEQ ID NO: 131) (SEQ ID NO: 135)(SEQ ID NO: 128) Repeat PD-1-right PD-1-right TALEN (SEQ ID NO: 132)(SEQ ID NO: 136)

In an embodiment, a method for expanding tumor infiltrating lymphocytes(TILs) into a therapeutic population of TILs comprises the steps of:

-   -   (a) activating a first population of TILs obtained from a tumor        resected from a patient using CD3 and CD28 activating beads or        antibodies for 1 to 5 days;    -   (b) gene-editing at least a portion of the first population of        TILs using electroporation of transcription activator-like        effector nucleases targeting PDCD1 in cytoporation medium to        obtain a second population of TILs;    -   (c) optionally incubating the second population of TILs, wherein        the incubation is performed at about 30-40° C. with about 5%        CO₂;    -   (d) performing a first expansion by culturing the second        population of TILs in a cell culture medium comprising IL-2, and        optionally OKT-3, to produce a third population of TILs, wherein        the first expansion is performed in a closed container providing        a first gas-permeable surface area, wherein the first expansion        is performed for about 6 to 9 days to obtain the third        population of TILs;    -   (e) performing a second expansion by supplementing the cell        culture medium of the third population of TILs with additional        IL-2, OKT-3, and antigen presenting cells (APCs), to produce a        fourth population of TILs, wherein the second expansion is        performed for about 9 to 11 days to obtain the fourth population        of TILs, wherein the fourth population of TILs is a therapeutic        population of TILs;    -   (f) harvesting the therapeutic population of TILs obtained from        step (e);    -   (g) transferring the harvested TIL population from step (e) to        an infusion bag, wherein the transfer from step (e) to (f)        occurs without opening the system; and    -   (h) wherein one or more of steps (a) to (g) are performed in a        closed, sterile system.

In an embodiment, a method for expanding tumor infiltrating lymphocytes(TILs) into a therapeutic population of TILs comprises the steps of:

-   -   (a) activating a first population of TILs obtained from a tumor        resected from a patient using CD3 and CD28 activating beads or        antibodies for 1 to 5 days;    -   (b) gene-editing at least a portion of the first population of        TILs using electroporation of transcription activator-like        effector nucleases targeting SEQ ID NO:128 in cytoporation        medium to obtain a second population of TILs;    -   (c) optionally incubating the second population of TILs, wherein        the incubation is performed at about 30-40° C. with about 5%        CO₂;    -   (d) performing a first expansion by culturing the second        population of TILs in a cell culture medium comprising IL-2, and        optionally OKT-3, to produce a third population of TILs, wherein        the first expansion is performed in a closed container providing        a first gas-permeable surface area, wherein the first expansion        is performed for about 6 to 9 days to obtain the third        population of TILs;    -   (e) performing a second expansion by supplementing the cell        culture medium of the third population of TILs with additional        IL-2, OKT-3, and antigen presenting cells (APCs), to produce a        fourth population of TILs, wherein the second expansion is        performed for about 9 to 11 days to obtain the fourth population        of TILs, wherein the fourth population of TILs is a therapeutic        population of TILs;    -   (f) harvesting the therapeutic population of TILs obtained from        step (e);    -   (g) transferring the harvested TIL population from step (e) to        an infusion bag, wherein the transfer from step (e) to (f)        occurs without opening the system; and    -   (h) wherein one or more of steps (a) to (g) are performed in a        closed, sterile system.

In an embodiment, a method for expanding tumor infiltrating lymphocytes(TILs) into a therapeutic population of TILs comprises the steps of:

-   -   (a) activating a first population of TILs obtained from a tumor        resected from a patient using CD3 and CD28 activating beads or        antibodies for 1 to 5 days;    -   (b) gene-editing at least a portion of the first population of        TILs using electroporation of transcription activator-like        effector nuclease mRNA according to SEQ ID NO: 135 and SEQ ID        NO:136 in cytoporation medium to obtain a second population of        TILs;    -   (c) optionally incubating the second population of TILs, wherein        the incubation is performed at about 30-40° C. with about 5%        CO₂;    -   (d) performing a first expansion by culturing the second        population of TILs in a cell culture medium comprising IL-2, and        optionally OKT-3, to produce a third population of TILs, wherein        the first expansion is performed in a closed container providing        a first gas-permeable surface area, wherein the first expansion        is performed for about 6 to 9 days to obtain the third        population of TILs;    -   (e) performing a second expansion by supplementing the cell        culture medium of the third population of TILs with additional        IL-2, OKT-3, and antigen presenting cells (APCs), to produce a        fourth population of TILs, wherein the second expansion is        performed for about 9 to 11 days to obtain the fourth population        of TILs, wherein the fourth population of TILs is a therapeutic        population of TILs;    -   (f) harvesting the therapeutic population of TILs obtained from        step (e);    -   (g) transferring the harvested TIL population from step (e) to        an infusion bag, wherein the transfer from step (e) to (f)        occurs without opening the system; and    -   (h) wherein one or more of steps (a) to (g) are performed in a        closed, sterile system.

Other non-limiting examples of genes that may be enhanced by permanentlygene-editing TILs via a TALE method include CCR2, CCR4, CCR5, CXCR2,CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-21, the NOTCH 1/2intracellular domain (ICD), and/or the NOTCH ligand mDLL1.

Examples of systems, methods, and compositions for altering theexpression of a target gene sequence by a TALE method, and which may beused in accordance with embodiments of the present invention, aredescribed in U.S. Pat. No. 8,586,526, which is incorporated by referenceherein. These disclosed examples include the use of a non-naturallyoccurring DNA-binding polypeptide that has two or more TALE-repeat unitscontaining a repeat RVD, an N-cap polypeptide made of residues of a TALEprotein, and a C-cap polypeptide made of a fragment of a full lengthC-terminus region of a TALE protein.

Examples of TALEN designs and design strategies, activity assessments,screening strategies, and methods that can be used to efficientlyperform TALEN-mediated gene integration and inactivation, and which maybe used in accordance with embodiments of the present invention, aredescribed in Valton, et al., Methods, 2014, 69, 151-170, which isincorporated by reference herein.

According to one embodiment, a method for expanding tumor infiltratinglymphocytes (TILs) into a therapeutic population of TILs comprises:

(a) obtaining a first population of TILs from a tumor resected from apatient by processing a tumor sample obtained from the patient intomultiple tumor fragments;

(b) adding the tumor fragments into a closed system;

(c) performing a first expansion by culturing the first population ofTILs in a cell culture medium comprising IL-2 and optionally comprisinga 4-1BB agonist antibody for about 2 to 5 days;

(d) adding OKT-3, to produce a second population of TILs, wherein thefirst expansion is performed in a closed container providing a firstgas-permeable surface area, wherein the first expansion is performed forabout 1 to 3 days to obtain the second population of TILs, wherein thesecond population of TILs is at least 50-fold greater in number than thefirst population of TILs, and wherein the transition from step (c) tostep (d) occurs without opening the system;

(e) performing a sterile electroporation step on the second populationof TILs, wherein the sterile electroporation step mediates the transferof at least one gene editor;

(f) resting the second population of TILs for about 1 day;

(g) performing a second expansion by supplementing the cell culturemedium of the second population of TILs with additional IL-2, optionallyOKT-3 antibody, optionally an OX40 antibody, and antigen presentingcells (APCs), to produce a third population of TILs, wherein the secondexpansion is performed for about 7 to 11 days to obtain the thirdpopulation of TILs, wherein the second expansion is performed in aclosed container providing a second gas-permeable surface area, andwherein the transition from step (f) to step (g) occurs without openingthe system;

(h) harvesting the therapeutic population of TILs obtained from step (g)to provide a harvested TIL population, wherein the transition from step(g) to step (h) occurs without opening the system, wherein the harvestedpopulation of TILs is a therapeutic population of TILs;

(i) transferring the harvested TIL population to an infusion bag,wherein the transfer from step (h) to (i) occurs without opening thesystem; and

(j) cryopreserving the harvested TIL population using adimethylsulfoxide-based cryopreservation medium,

wherein the electroporation step comprises the delivery of a TALEnuclease system for modulating the expression of at least one protein.

According to one embodiment, a method for expanding tumor infiltratinglymphocytes (TILs) into a therapeutic population of TILs comprises:

(a) obtaining a first population of TILs from a tumor resected from apatient by processing a tumor sample obtained from the patient intomultiple tumor fragments;

(b) adding the tumor fragments into a closed system;

(c) performing a first expansion by culturing the first population ofTILs in a cell culture medium comprising IL-2 and optionally comprisinga 4-1BB agonist antibody for about 2 to 5 days;

(d) adding OKT-3, to produce a second population of TILs, wherein thefirst expansion is performed in a closed container providing a firstgas-permeable surface area, wherein the first expansion is performed forabout 1 to 3 days to obtain the second population of TILs, wherein thesecond population of TILs is at least 50-fold greater in number than thefirst population of TILs, and wherein the transition from step (c) tostep (d) occurs without opening the system;

(e) performing a sterile electroporation step on the second populationof TILs, wherein the sterile electroporation step mediates the transferof at least one gene editor;

(f) resting the second population of TILs for about 1 day;

(g) performing a second expansion by supplementing the cell culturemedium of the second population of TILs with additional IL-2, optionallyOKT-3 antibody, optionally an OX40 antibody, and antigen presentingcells (APCs), to produce a third population of TILs, wherein the secondexpansion is performed for about 7 to 11 days to obtain the thirdpopulation of TILs, wherein the second expansion is performed in aclosed container providing a second gas-permeable surface area, andwherein the transition from step (f) to step (g) occurs without openingthe system;

(h) harvesting the therapeutic population of TILs obtained from step (g)to provide a harvested TIL population, wherein the transition from step(g) to step (h) occurs without opening the system, wherein the harvestedpopulation of TILs is a therapeutic population of TILs;

(i) transferring the harvested TIL population to an infusion bag,wherein the transfer from step (h) to (i) occurs without opening thesystem; and

(j) cryopreserving the harvested TIL population using adimethylsulfoxide-based cryopreservation medium,

wherein the electroporation step comprises the delivery of a TALEnuclease system for suppressing the expression of PD-1 and LAG-3.

3. Zinc Finger Methods

A method for expanding TILs into a therapeutic population may be carriedout in accordance with any embodiment of the methods described herein(e.g., process 2A) or as described in PCT/US2017/058610,PCT/US2018/012605, or PCT/US2018/012633, wherein the method furthercomprises gene-editing at least a portion of the TILs by a zinc fingeror zinc finger nuclease method. According to particular embodiments, theuse of a zinc finger method during the TIL expansion process causesexpression of one or more immune checkpoint genes to be silenced orreduced in at least a portion of the therapeutic population of TILs.Alternatively, the use of a zinc finger method during the TIL expansionprocess causes expression of one or more immune checkpoint genes to beenhanced in at least a portion of the therapeutic population of TILs.

An individual zinc finger contains approximately 30 amino acids in aconserved ββα configuration. Several amino acids on the surface of theα-helix typically contact 3 bp in the major groove of DNA, with varyinglevels of selectivity. Zinc fingers have two protein domains. The firstdomain is the DNA binding domain, which includes eukaryotictranscription factors and contain the zinc finger. The second domain isthe nuclease domain, which includes the FokI restriction enzyme and isresponsible for the catalytic cleavage of DNA.

The DNA-binding domains of individual ZFNs typically contain betweenthree and six individual zinc finger repeats and can each recognizebetween 9 and 18 base pairs. If the zinc finger domains are specific fortheir intended target site then even a pair of 3-finger ZFNs thatrecognize a total of 18 base pairs can, in theory, target a single locusin a mammalian genome. One method to generate new zinc-finger arrays isto combine smaller zinc-finger “modules” of known specificity. The mostcommon modular assembly process involves combining three separate zincfingers that can each recognize a 3 base pair DNA sequence to generate a3-finger array that can recognize a 9 base pair target site.Alternatively, selection-based approaches, such as oligomerized poolengineering (OPEN) can be used to select for new zinc-finger arrays fromrandomized libraries that take into consideration context-dependentinteractions between neighboring fingers. Engineered zinc fingers areavailable commercially; Sangamo Biosciences (Richmond, Calif., USA) hasdeveloped a propriety platform (CompoZr®) for zinc-finger constructionin partnership with Sigma-Aldrich (St. Louis, Mo., USA).

Non-limiting examples of genes that may be silenced or inhibited bypermanently gene-editing TILs via a zinc finger method include PD-1,CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB,PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7,SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6,CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA,IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1,BATF, GUCY1A2, GUCY1A3, GUCY1B2, and GUCY1B3.

Non-limiting examples of genes that may be enhanced by permanentlygene-editing TILs via a zinc finger method include CCR2, CCR4, CCR5,CXCR2, CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-21, the NOTCH1/2 intracellular domain (ICD), and/or the NOTCH ligand mDLL1.

Examples of systems, methods, and compositions for altering theexpression of a target gene sequence by a zinc finger method, which maybe used in accordance with embodiments of the present invention, aredescribed in U.S. Pat. Nos. 6,534,261, 6,607,882, 6,746,838, 6,794,136,6,824,978, 6,866,997, 6,933,113, 6,979,539, 7,013,219, 7,030,215,7,220,719, 7,241,573, 7,241,574, 7,585,849, 7,595,376, 6,903,185, and6,479,626, which are incorporated by reference herein.

Other examples of systems, methods, and compositions for altering theexpression of a target gene sequence by a zinc finger method, which maybe used in accordance with embodiments of the present invention, aredescribed in Beane, et al., Mol. Therapy, 2015, 23 1380-1390, thedisclosure of which is incorporated by reference herein.

According to one embodiment, a method for expanding tumor infiltratinglymphocytes (TILs) into a therapeutic population of TILs comprises:

(a) obtaining a first population of TILs from a tumor resected from apatient by processing a tumor sample obtained from the patient intomultiple tumor fragments;

(b) adding the tumor fragments into a closed system;

(c) performing a first expansion by culturing the first population ofTILs in a cell culture medium comprising IL-2 and optionally comprisinga 4-1BB agonist antibody for about 2 to 5 days;

(d) adding OKT-3, to produce a second population of TILs, wherein thefirst expansion is performed in a closed container providing a firstgas-permeable surface area, wherein the first expansion is performed forabout 1 to 3 days to obtain the second population of TILs, wherein thesecond population of TILs is at least 50-fold greater in number than thefirst population of TILs, and wherein the transition from step (c) tostep (d) occurs without opening the system;

(e) performing a sterile electroporation step on the second populationof TILs, wherein the sterile electroporation step mediates the transferof at least one gene editor;

(f) resting the second population of TILs for about 1 day;

(g) performing a second expansion by supplementing the cell culturemedium of the second population of TILs with additional IL-2, optionallyOKT-3 antibody, optionally an OX40 antibody, and antigen presentingcells (APCs), to produce a third population of TILs, wherein the secondexpansion is performed for about 7 to 11 days to obtain the thirdpopulation of TILs, wherein the second expansion is performed in aclosed container providing a second gas-permeable surface area, andwherein the transition from step (f) to step (g) occurs without openingthe system;

(h) harvesting the therapeutic population of TILs obtained from step (g)to provide a harvested TIL population, wherein the transition from step(g) to step (h) occurs without opening the system, wherein the harvestedpopulation of TILs is a therapeutic population of TILs;

(i) transferring the harvested TIL population to an infusion bag,wherein the transfer from step (h) to (i) occurs without opening thesystem; and

(j) cryopreserving the harvested TIL population using adimethylsulfoxide-based cryopreservation medium, wherein theelectroporation step comprises the delivery of a zinc finger nucleasesystem for modulating the expression of at least one protein.

According to one embodiment, a method for expanding tumor infiltratinglymphocytes (TILs) into a therapeutic population of TILs comprises:

(a) obtaining a first population of TILs from a tumor resected from apatient by processing a tumor sample obtained from the patient intomultiple tumor fragments;

(b) adding the tumor fragments into a closed system;

(c) performing a first expansion by culturing the first population ofTILs in a cell culture medium comprising IL-2 and optionally comprisinga 4-1BB agonist antibody for about 2 to 5 days;

(d) adding OKT-3, to produce a second population of TILs, wherein thefirst expansion is performed in a closed container providing a firstgas-permeable surface area, wherein the first expansion is performed forabout 1 to 3 days to obtain the second population of TILs, wherein thesecond population of TILs is at least 50-fold greater in number than thefirst population of TILs, and wherein the transition from step (c) tostep (d) occurs without opening the system;

(e) performing a sterile electroporation step on the second populationof TILs, wherein the sterile electroporation step mediates the transferof at least one gene editor;

(f) resting the second population of TILs for about 1 day;

(g) performing a second expansion by supplementing the cell culturemedium of the second population of TILs with additional IL-2, optionallyOKT-3 antibody, optionally an OX40 antibody, and antigen presentingcells (APCs), to produce a third population of TILs, wherein the secondexpansion is performed for about 7 to 11 days to obtain the thirdpopulation of TILs, wherein the second expansion is performed in aclosed container providing a second gas-permeable surface area, andwherein the transition from step (f) to step (g) occurs without openingthe system;

(h) harvesting the therapeutic population of TILs obtained from step (g)to provide a harvested TIL population, wherein the transition from step(g) to step (h) occurs without opening the system, wherein the harvestedpopulation of TILs is a therapeutic population of TILs;

(i) transferring the harvested TIL population to an infusion bag,wherein the transfer from step (h) to (i) occurs without opening thesystem; and

(j) cryopreserving the harvested TIL population using adimethylsulfoxide-based cryopreservation medium, wherein theelectroporation step comprises the delivery of a zinc finger nucleasesystem for suppressing the expression of PD-1 and LAG-3.

IV. TIL Manufacturing Processes

An exemplary TIL process known as process 2A containing some of thesefeatures is depicted in FIG. 1, and some of the advantages of thisembodiment of the present invention over process 1C are described inFIG. 2, as does FIG. 14. Process 1C is shown for comparison in FIG. 3.Two alternative timelines for TIL therapy based on process 2A are shownin FIG. 4 (higher cell counts) and FIG. 5 (lower cell counts). Anembodiment of process 2A is shown in FIG. 6 as well as FIG. 9. FIGS. 13and 14 further provides an exemplary 2A process compared to an exemplary1C process.

As discussed herein, the present invention can include a step relatingto the restimulation of cryopreserved TILs to increase their metabolicactivity and thus relative health prior to transplant into a patient,and methods of testing said metabolic health. As generally outlinedherein, TILs are generally taken from a patient sample and manipulatedto expand their number prior to transplant into a patient. In someembodiments, the TILs may be optionally genetically manipulated asdiscussed below.

In some embodiments, the TILs may be cryopreserved. Once thawed, theymay also be restimulated to increase their metabolism prior to infusioninto a patient.

In some embodiments, the first expansion (including processes referredto as the preREP as well as processes shown in FIG. 9 as Step A) isshortened to 3 to 14 days and the second expansion (including processesreferred to as the REP as well as processes shown in FIG. 9 as Step B)is shorted to 7 to 14 days, as discussed in detail below as well as inthe examples and figures. In some embodiments, the first expansion (forexample, an expansion described as Step B in FIG. 9) is shortened to 11days and the second expansion (for example, an expansion as described inStep D in FIG. 9) is shortened to 11 days, as discussed in the Examplesand shown in FIGS. 4, 5 and 27. In some embodiments, the combination ofthe first expansion and second expansion (for example, expansionsdescribed as Step B and Step D in FIG. 9) is shortened to 22 days, asdiscussed in detail below and in the examples and figures.

The “Step” Designations A, B, C, etc., below are in reference to FIG. 9and in reference to certain embodiments described herein. The orderingof the Steps below and in FIG. 9 is exemplary and any combination ororder of steps, as well as additional steps, repetition of steps, and/oromission of steps is contemplated by the present application and themethods disclosed herein.

A. STEP A: Obtain Patient Tumor Sample

In general, TILs are initially obtained from a patient tumor sample(“primary TILs”) and then expanded into a larger population for furthermanipulation as described herein, optionally cryopreserved, restimulatedas outlined herein and optionally evaluated for phenotype and metabolicparameters as an indication of TIL health.

A patient tumor sample may be obtained using methods known in the art,generally via surgical resection, needle biopsy or other means forobtaining a sample that contains a mixture of tumor and TIL cells. Ingeneral, the tumor sample may be from any solid tumor, including primarytumors, invasive tumors or metastatic tumors. The tumor sample may alsobe a liquid tumor, such as a tumor obtained from a hematologicalmalignancy. The solid tumor may be of any cancer type, including, butnot limited to, breast, pancreatic, prostate, colorectal, lung, brain,renal, stomach, and skin (including but not limited to squamous cellcarcinoma, basal cell carcinoma, and melanoma). In some embodiments,useful TILs are obtained from malignant melanoma tumors, as these havebeen reported to have particularly high levels of TILs.

The term “solid tumor” refers to an abnormal mass of tissue that usuallydoes not contain cysts or liquid areas. Solid tumors may be benign ormalignant. The term “solid tumor cancer” refers to malignant,neoplastic, or cancerous solid tumors. Solid tumor cancers include, butare not limited to, sarcomas, carcinomas, and lymphomas, such as cancersof the lung, breast, triple negative breast cancer, prostate, colon,rectum, and bladder. In some embodiments, the cancer is selected fromcervical cancer, head and neck cancer (including, for example, head andneck squamous cell carcinoma (HNSCC)) glioblastoma, ovarian cancer,sarcoma, pancreatic cancer, bladder cancer, breast cancer, triplenegative breast cancer, and non-small cell lung carcinoma. The tissuestructure of solid tumors includes interdependent tissue compartmentsincluding the parenchyma (cancer cells) and the supporting stromal cellsin which the cancer cells are dispersed and which may provide asupporting microenvironment.

The term “hematological malignancy” refers to mammalian cancers andtumors of the hematopoietic and lymphoid tissues, including but notlimited to tissues of the blood, bone marrow, lymph nodes, and lymphaticsystem. Hematological malignancies are also referred to as “liquidtumors.” Hematological malignancies include, but are not limited to,acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL),small lymphocytic lymphoma (SLL), acute myelogenous leukemia (AML),chronic myelogenous leukemia (CML), acute monocytic leukemia (AMoL),Hodgkin's lymphoma, and non-Hodgkin's lymphomas. The term “B cellhematological malignancy” refers to hematological malignancies thataffect B cells.

Once obtained, the tumor sample is generally fragmented using sharpdissection into small pieces of between 1 to about 8 mm³, with fromabout 2-3 mm³ being particularly useful. The TILs are cultured fromthese fragments using enzymatic tumor digests. Such tumor digests may beproduced by incubation in enzymatic media (e.g., Roswell Park MemorialInstitute (RPMI) 1640 buffer, 2 mM glutamate, 10 mcg/mL gentamicin, 30units/mL of DNase and 1.0 mg/mL of collagenase) followed by mechanicaldissociation (e.g., using a tissue dissociator). Tumor digests may beproduced by placing the tumor in enzymatic media and mechanicallydissociating the tumor for approximately 1 minute, followed byincubation for 30 minutes at 37° C. in 5% CO₂, followed by repeatedcycles of mechanical dissociation and incubation under the foregoingconditions until only small tissue pieces are present. At the end ofthis process, if the cell suspension contains a large number of redblood cells or dead cells, a density gradient separation using FICOLLbranched hydrophilic polysaccharide may be performed to remove thesecells. Alternative methods known in the art may be used, such as thosedescribed in U.S. Patent Application Publication No. 2012/0244133 A1,the disclosure of which is incorporated by reference herein. Any of theforegoing methods may be used in any of the embodiments described hereinfor methods of expanding TILs or methods treating a cancer.

In general, the harvested cell suspension is called a “primary cellpopulation” or a “freshly harvested” cell population.

In some embodiments, fragmentation includes physical fragmentation,including for example, dissection as well as digestion. In someembodiments, the fragmentation is physical fragmentation. In someembodiments, the fragmentation is dissection. In some embodiments, thefragmentation is by digestion. In some embodiments, TILs can beinitially cultured from enzymatic tumor digests and tumor fragmentsobtained from patients. In an embodiment, TILs can be initially culturedfrom enzymatic tumor digests and tumor fragments obtained from patients.

In some embodiments, where the tumor is a solid tumor, the tumorundergoes physical fragmentation after the tumor sample is obtained in,for example, Step A (as provided in FIG. 9). In some embodiments, thefragmentation occurs before cryopreservation. In some embodiments, thefragmentation occurs after cryopreservation. In some embodiments, thefragmentation occurs after obtaining the tumor and in the absence of anycryopreservation. In some embodiments, the tumor is fragmented and 10,20, 30, 40 or more fragments or pieces are placed in each container forthe first expansion. In some embodiments, the tumor is fragmented and 30or 40 fragments or pieces are placed in each container for the firstexpansion. In some embodiments, the tumor is fragmented and 40 fragmentsor pieces are placed in each container for the first expansion. In someembodiments, the multiple fragments comprise about 4 to about 50fragments, wherein each fragment has a volume of about 27 mm³. In someembodiments, the multiple fragments comprise about 30 to about 60fragments with a total volume of about 1300 mm³ to about 1500 mm³. Insome embodiments, the multiple fragments comprise about 50 fragmentswith a total volume of about 1350 mm³. In some embodiments, the multiplefragments comprise about 50 fragments with a total mass of about 1 gramto about 1.5 grams. In some embodiments, the multiple fragments compriseabout 4 fragments.

In some embodiments, the TILs are obtained from tumor fragments. In someembodiments, the tumor fragment is obtained by sharp dissection. In someembodiments, the tumor fragment is between about 1 mm³ and 10 mm³. Insome embodiments, the tumor fragment is between about 1 mm³ and 8 mm³.In some embodiments, the tumor fragment is about 1 mm³. In someembodiments, the tumor fragment is about 2 mm³. In some embodiments, thetumor fragment is about 3 mm³. In some embodiments, the tumor fragmentis about 4 mm³. In some embodiments, the tumor fragment is about 5 mm³.In some embodiments, the tumor fragment is about 6 mm³. In someembodiments, the tumor fragment is about 7 mm³. In some embodiments, thetumor fragment is about 8 mm³. In some embodiments, the tumor fragmentis about 9 mm³. In some embodiments, the tumor fragment is about 10 mm³.

In some embodiments, the TILs are obtained from tumor digests. In someembodiments, tumor digests were generated by incubation in enzyme media,for example but not limited to RPMI 1640, 2 mM GlutaMAX, 10 mg/mLgentamicin, 30 U/mL DNase, and 1.0 mg/mL collagenase, followed bymechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, Calif.).After placing the tumor in enzyme media, the tumor can be mechanicallydissociated for approximately 1 minute. The solution can then beincubated for 30 minutes at 37° C. in 5% CO₂ and it then mechanicallydisrupted again for approximately 1 minute. After being incubated againfor 30 minutes at 37° C. in 5% CO₂, the tumor can be mechanicallydisrupted a third time for approximately 1 minute. In some embodiments,after the third mechanical disruption if large pieces of tissue werepresent, 1 or 2 additional mechanical dissociations were applied to thesample, with or without 30 additional minutes of incubation at 37° C. in5% CO₂. In some embodiments, at the end of the final incubation if thecell suspension contained a large number of red blood cells or deadcells, a density gradient separation using Ficoll can be performed toremove these cells.

In some embodiments, the harvested cell suspension prior to the firstexpansion step is called a “primary cell population” or a “freshlyharvested” cell population.

In some embodiments, cells can be optionally frozen after sample harvestand stored frozen prior to entry into the expansion described in Step B,which is described in further detail below, as well as exemplified inFIG. 9.

B. STEP B: First Expansion

1. Young TILs

In some embodiments, the present methods provide for obtaining youngTILs, which are capable of increased replication cycles uponadministration to a subject/patient and as such may provide additionaltherapeutic benefits over older TILs (i.e., TILs which have furtherundergone more rounds of replication prior to administration to asubject/patient). Features of young TILs have been described in theliterature, for example Donia, at al., Scandinavian Journal ofImmunology, 75:157-167 (2012); Dudley et al., Clin Cancer Res,16:6122-6131 (2010); Huang et al., J Immunother, 28(3):258-267 (2005);Besser et al., Clin Cancer Res, 19(17):OF1-OF9 (2013); Besser et al., JImmunother 32:415-423 (2009); Robbins, et al., J Immunol 2004;173:7125-7130; Shen et al., J Immunother, 30:123-129 (2007); Zhou, etal., J Immunother, 28:53-62 (2005); and Tran, et al., J Immunother,31:742-751 (2008), all of which are incorporated herein by reference intheir entireties.

The diverse antigen receptors of T and B lymphocytes are produced bysomatic recombination of a limited, but large number of gene segments.These gene segments: V (variable), D (diversity), J (joining), and C(constant), determine the binding specificity and downstreamapplications of immunoglobulins and T-cell receptors (TCRs). The presentinvention provides a method for generating TILs which exhibit andincrease the T-cell repertoire diversity. In some embodiments, the TILsobtained by the present method exhibit an increase in the T-cellrepertoire diversity. In some embodiments, the TILs obtained by thepresent method exhibit an increase in the T-cell repertoire diversity ascompared to freshly harvested TILs and/or TILs prepared using othermethods than those provide herein including for example, methods otherthan those embodied in FIG. 9. In some embodiments, the TILs obtained bythe present method exhibit an increase in the T-cell repertoirediversity as compared to freshly harvested TILs and/or TILs preparedusing methods referred to as process 1C, as exemplified in FIG. 13. Insome embodiments, the TILs obtained in the first expansion exhibit anincrease in the T-cell repertoire diversity. In some embodiments, theincrease in diversity is an increase in the immunoglobulin diversityand/or the T-cell receptor diversity. In some embodiments, the diversityis in the immunoglobulin is in the immunoglobulin heavy chain. In someembodiments, the diversity is in the immunoglobulin is in theimmunoglobulin light chain. In some embodiments, the diversity is in theT-cell receptor. In some embodiments, the diversity is in one of theT-cell receptors selected from the group consisting of alpha, beta,gamma, and delta receptors. In some embodiments, there is an increase inthe expression of T-cell receptor (TCR) alpha and/or beta. In someembodiments, there is an increase in the expression of T-cell receptor(TCR) alpha. In some embodiments, there is an increase in the expressionof T-cell receptor (TCR) beta. In some embodiments, there is an increasein the expression of TCRab (i.e., TCRα/β).

After dissection or digestion of tumor fragments, for example such asdescribed in Step A of FIG. 9, the resulting cells are cultured in serumcontaining IL-2 under conditions that favor the growth of TILs overtumor and other cells. In some embodiments, the tumor digests areincubated in 2 mL wells in media comprising inactivated human AB serumwith 6000 IU/mL of IL-2. This primary cell population is cultured for aperiod of days, generally from 3 to 14 days, resulting in a bulk TILpopulation, generally about 1×10⁸ bulk TIL cells. In some embodiments,this primary cell population is cultured for a period of 7 to 14 days,resulting in a bulk TIL population, generally about 1×10⁸ bulk TILcells. In some embodiments, this primary cell population is cultured fora period of 10 to 14 days, resulting in a bulk TIL population, generallyabout 1×10⁸ bulk TIL cells. In some embodiments, this primary cellpopulation is cultured for a period of about 11 days, resulting in abulk TIL population, generally about 1×10⁸ bulk TIL cells.

In a preferred embodiment, expansion of TILs may be performed using aninitial bulk TIL expansion step (for example such as those described inStep B of FIG. 9, which can include processes referred to as pre-REP) asdescribed below and herein, followed by a second expansion (Step D,including processes referred to as rapid expansion protocol (REP) steps)as described below under Step D and herein, followed by optionalcryopreservation, and followed by a second Step D (including processesreferred to as restimulation REP steps) as described below and herein.The TILs obtained from this process may be optionally characterized forphenotypic characteristics and metabolic parameters as described herein.

In embodiments where TIL cultures are initiated in 24-well plates, forexample, using Costar 24-well cell culture cluster, flat bottom (CorningIncorporated, Corning, N.Y., each well can be seeded with 1×10⁶ tumordigest cells or one tumor fragment in 2 mL of complete medium (CM) withIL-2 (6000 IU/mL; Chiron Corp., Emeryville, Calif.). In someembodiments, the tumor fragment is between about 1 mm³ and 10 mm³.

In some embodiments, the first expansion culture medium is referred toas “CM”, an abbreviation for culture media. In some embodiments, CM forStep B consists of RPMI 1640 with GlutaMAX, supplemented with 10% humanAB serum, 25 mM Hepes, and 10 mg/mL gentamicin. In embodiments wherecultures are initiated in gas-permeable flasks with a 40 mL capacity anda 10 cm² gas-permeable silicon bottom (for example, G-Rex10; Wilson WolfManufacturing, New Brighton, Minn.) (FIG. 1), each flask was loaded with10-40×10⁶ viable tumor digest cells or 5-30 tumor fragments in 10-40 mLof CM with IL-2. Both the G-Rex10 and 24-well plates were incubated in ahumidified incubator at 37° C. in 5% CO₂ and 5 days after cultureinitiation, half the media was removed and replaced with fresh CM andIL-2 and after day 5, half the media was changed every 2-3 days.

After preparation of the tumor fragments, the resulting cells (i.e.,fragments) are cultured in serum containing IL-2 under conditions thatfavor the growth of TILs over tumor and other cells. In someembodiments, the tumor digests are incubated in 2 mL wells in mediacomprising inactivated human AB serum (or, in some cases, as outlinedherein, in the presence of aAPC cell population) with 6000 IU/mL ofIL-2. This primary cell population is cultured for a period of days,generally from 10 to 14 days, resulting in a bulk TIL population,generally about 1×10⁸ bulk TIL cells. In some embodiments, the growthmedia during the first expansion comprises IL-2 or a variant thereof. Insome embodiments, the IL is recombinant human IL-2 (rhIL-2). In someembodiments the IL-2 stock solution has a specific activity of 20-30×10⁶IU/mg for a 1 mg vial. In some embodiments the IL-2 stock solution has aspecific activity of 20×10⁶ IU/mg for a 1 mg vial. In some embodimentsthe IL-2 stock solution has a specific activity of 25×10⁶ IU/mg for a 1mg vial. In some embodiments the IL-2 stock solution has a specificactivity of 30×10⁶ IU/mg for a 1 mg vial. In some embodiments, the IL-2stock solution has a final concentration of 4-8×10⁶ IU/mg of IL-2. Insome embodiments, the IL-2 stock solution has a final concentration of5-7×10⁶ IU/mg of IL-2. In some embodiments, the IL-2 stock solution hasa final concentration of 6×10⁶ IU/mg of IL-2. In some embodiments, theIL-2 stock solution is prepare as described in Example 4. In someembodiments, the first expansion culture media comprises about 10,000IU/mL of IL-2, about 9,000 IU/mL of IL-2, about 8,000 IU/mL of IL-2,about 7,000 IU/mL of IL-2, about 6000 IU/mL of IL-2 or about 5,000 IU/mLof IL-2. In some embodiments, the first expansion culture mediacomprises about 9,000 IU/mL of IL-2 to about 5,000 IU/mL of IL-2. Insome embodiments, the first expansion culture media comprises about8,000 IU/mL of IL-2 to about 6,000 IU/mL of IL-2. In some embodiments,the first expansion culture media comprises about 7,000 IU/mL of IL-2 toabout 6,000 IU/mL of IL-2. In some embodiments, the first expansionculture media comprises about 6,000 IU/mL of IL-2. In an embodiment, thecell culture medium further comprises IL-2. In some embodiments, thecell culture medium comprises about 3000 IU/mL of IL-2. In anembodiment, the cell culture medium further comprises IL-2. In apreferred embodiment, the cell culture medium comprises about 3000 IU/mLof IL-2. In an embodiment, the cell culture medium comprises about 1000IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000IU/mL, about 3500 IU/mL, about 4000 IU/mL, about 4500 IU/mL, about 5000IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000IU/mL, about 7500 IU/mL, or about 8000 IU/mL of IL-2. In an embodiment,the cell culture medium comprises between 1000 and 2000 IU/mL, between2000 and 3000 IU/mL, between 3000 and 4000 IU/mL, between 4000 and 5000IU/mL, between 5000 and 6000 IU/mL, between 6000 and 7000 IU/mL, between7000 and 8000 IU/mL, or about 8000 IU/mL of IL-2.

In some embodiments, first expansion culture media comprises about 500IU/mL of IL-15, about 400 IU/mL of IL-15, about 300 IU/mL of IL-15,about 200 IU/mL of IL-15, about 180 IU/mL of IL-15, about 160 IU/mL ofIL-15, about 140 IU/mL of IL-15, about 120 IU/mL of IL-15, or about 100IU/mL of IL-15. In some embodiments, the first expansion culture mediacomprises about 500 IU/mL of IL-15 to about 100 IU/mL of IL-15. In someembodiments, the first expansion culture media comprises about 400 IU/mLof IL-15 to about 100 IU/mL of IL-15. In some embodiments, the firstexpansion culture media comprises about 300 IU/mL of IL-15 to about 100IU/mL of IL-15. In some embodiments, the first expansion culture mediacomprises about 200 IU/mL of IL-15. In some embodiments, the cellculture medium comprises about 180 IU/mL of IL-15. In an embodiment, thecell culture medium further comprises IL-15. In a preferred embodiment,the cell culture medium comprises about 180 IU/mL of IL-15.

In some embodiments, first expansion culture media comprises about 20IU/mL of IL-21, about 15 IU/mL of IL-21, about 12 IU/mL of IL-21, about10 IU/mL of IL-21, about 5 IU/mL of IL-21, about 4 IU/mL of IL-21, about3 IU/mL of IL-21, about 2 IU/mL of IL-21, about 1 IU/mL of IL-21, orabout 0.5 IU/mL of IL-21. In some embodiments, the first expansionculture media comprises about 20 IU/mL of IL-21 to about 0.5 IU/mL ofIL-21. In some embodiments, the first expansion culture media comprisesabout 15 IU/mL of IL-21 to about 0.5 IU/mL of IL-21. In someembodiments, the first expansion culture media comprises about 12 IU/mLof IL-21 to about 0.5 IU/mL of IL-21. In some embodiments, the firstexpansion culture media comprises about 10 IU/mL of IL-21 to about 0.5IU/mL of IL-21. In some embodiments, the first expansion culture mediacomprises about 5 IU/mL of IL-21 to about 1 IU/mL of IL-21. In someembodiments, the first expansion culture media comprises about 2 IU/mLof IL-21. In some embodiments, the cell culture medium comprises about 1IU/mL of IL-21. In some embodiments, the cell culture medium comprisesabout 0.5 IU/mL of IL-21. In an embodiment, the cell culture mediumfurther comprises IL-21. In a preferred embodiment, the cell culturemedium comprises about 1 IU/mL of IL-21.

In an embodiment, the cell culture medium comprises OKT-3 antibody. TheOKT-3 antibody may be present in the cell culture medium beginning onday 0 of the REP (i.e., the start day of the REP) and/or day 0 of thesecond expansion (i.e., the start day of the second expansion). In someembodiments, the cell culture medium comprises about 30 ng/mL of OKT-3antibody. In an embodiment, the cell culture medium comprises about 0.1ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about 5 ng/mL,about 7.5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL,about 100 ng/mL, about 200 ng/mL, about 500 ng/mL, and about 1 μg/mL ofOKT-3 antibody. In an embodiment, the cell culture medium comprisesbetween 0.1 ng/mL and 1 ng/mL, between 1 ng/mL and 5 ng/mL, between 5ng/mL and 10 ng/mL, between 10 ng/mL and 20 ng/mL, between 20 ng/mL and30 ng/mL, between 30 ng/mL and 40 ng/mL, between 40 ng/mL and 50 ng/mL,and between 50 ng/mL and 100 ng/mL of OKT-3 antibody. In someembodiments, the cell culture medium does not comprise OKT-3 antibody.

In some embodiments, the first expansion culture medium is referred toas “CM”, an abbreviation for culture media. In some embodiments, it isreferred to as CM1 (culture medium 1). In some embodiments, CM consistsof RPMI 1640 with GlutaMAX, supplemented with 10% human AB serum, 25 mMHepes, and 10 mg/mL gentamicin. In embodiments where cultures areinitiated in gas-permeable flasks with a 40 mL capacity and a 10 cm²gas-permeable silicon bottom (for example, G-Rex10; Wilson WolfManufacturing, New Brighton, Minn.) (FIG. 1), each flask was loaded with10-40×10⁶ viable tumor digest cells or 5-30 tumor fragments in 10-40 mLof CM with IL-2. Both the G-Rex10 and 24-well plates were incubated in ahumidified incubator at 37° C. in 5% CO₂ and 5 days after cultureinitiation, half the media was removed and replaced with fresh CM andIL-2 and after day 5, half the media was changed every 2-3 days. In someembodiments, the CM is the CM1 described in the Examples, see, Example5. In some embodiments, the first expansion occurs in an initial cellculture medium or a first cell culture medium. In some embodiments, theinitial cell culture medium or the first cell culture medium comprisesIL-2.

In some embodiments, the first expansion (including processes such asfor example those described in Step B of FIG. 9, which can include thosesometimes referred to as the pre-REP) process is shortened to 3-14 days,as discussed in the examples and figures. In some embodiments, the firstexpansion (including processes such as for example those described inStep B of FIG. 9, which can include those sometimes referred to as thepre-REP) is shortened to 7 to 14 days, as discussed in the Examples andshown in FIGS. 4 and 5, as well as including for example, an expansionas described in Step B of FIG. 9. In some embodiments, the firstexpansion of Step B is shortened to 10-14 days, as discussed in theExamples and shown in FIGS. 4 and 5. In some embodiments, the firstexpansion is shortened to 11 days, as discussed in the Examples andshown in FIGS. 4 and 5, as well as including for example, an expansionas described in Step B of FIG. 9.

In some embodiments, the first TIL expansion can proceed for 1 day, 2days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days,11 days, 12 days, 13 days, or 14 days. In some embodiments, the firstTIL expansion can proceed for 1 day to 14 days. In some embodiments, thefirst TIL expansion can proceed for 2 days to 14 days. In someembodiments, the first TIL expansion can proceed for 3 days to 14 days.In some embodiments, the first TIL expansion can proceed for 4 days to14 days. In some embodiments, the first TIL expansion can proceed for 5days to 14 days. In some embodiments, the first TIL expansion canproceed for 6 days to 14 days. In some embodiments, the first TILexpansion can proceed for 7 days to 14 days. In some embodiments, thefirst TIL expansion can proceed for 8 days to 14 days. In someembodiments, the first TIL expansion can proceed for 9 days to 14 days.In some embodiments, the first TIL expansion can proceed for 10 days to14 days. In some embodiments, the first TIL expansion can proceed for 11days to 14 days. In some embodiments, the first TIL expansion canproceed for 12 days to 14 days. In some embodiments, the first TILexpansion can proceed for 13 days to 14 days. In some embodiments, thefirst TIL expansion can proceed for 14 days. In some embodiments, thefirst TIL expansion can proceed for 1 day to 11 days. In someembodiments, the first TIL expansion can proceed for 2 days to 11 days.In some embodiments, the first TIL expansion can proceed for 3 days to11 days. In some embodiments, the first TIL expansion can proceed for 4days to 11 days. In some embodiments, the first TIL expansion canproceed for 5 days to 11 days. In some embodiments, the first TILexpansion can proceed for 6 days to 11 days. In some embodiments, thefirst TIL expansion can proceed for 7 days to 11 days. In someembodiments, the first TIL expansion can proceed for 8 days to 11 days.In some embodiments, the first TIL expansion can proceed for 9 days to11 days. In some embodiments, the first TIL expansion can proceed for 10days to 11 days. In some embodiments, the first TIL expansion canproceed for 11 days.

In some embodiments, a combination of IL-2, IL-7, IL-15, and/or IL-21are employed as a combination during the first expansion. In someembodiments, IL-2, IL-7, IL-15, and/or IL-21 as well as any combinationsthereof can be included during the first expansion, including forexample during a Step B processes according to FIG. 9, as well asdescribed herein. In some embodiments, a combination of IL-2, IL-15, andIL-21 are employed as a combination during the first expansion. In someembodiments, IL-2, IL-15, and IL-21 as well as any combinations thereofcan be included during Step B processes according to FIG. 9 and asdescribed herein.

In some embodiments, the first expansion (including processes referredto as the pre-REP; for example, Step B according to FIG. 9) process isshortened to 3 to 14 days, as discussed in the examples and figures. Insome embodiments, the first expansion of Step B is shortened to 7 to 14days, as discussed in the Examples and shown in FIGS. 4 and 5. In someembodiments, the first expansion of Step B is shortened to 10 to 14days, as discussed in the Examples and shown in FIGS. 4, 5, and 9. Insome embodiments, the first expansion is shortened to 11 days, asdiscussed in the Examples and shown in FIGS. 4, 5, and 9.

In some embodiments, the first expansion, for example, Step B accordingto FIG. 9, is performed in a closed system bioreactor. In someembodiments, a closed system is employed for the TIL expansion, asdescribed herein. In some embodiments, a single bioreactor is employed.In some embodiments, the single bioreactor employed is for example aG-REX-10 or a G-REX-100. In some embodiments, the closed systembioreactor is a single bioreactor.

In some embodiments, the first expansion is performed using anadditional 4-1BB agonist antibody added to the cell culture medium atthe start of the expansion, using any of the 4-1BB agonist antibodiesdescribed hereinafter.

C. STEP C: First Expansion to Second Expansion Transition

In some cases, the bulk TIL population obtained from the firstexpansion, including for example the TIL population obtained from forexample, Step B as indicated in FIG. 9, can be cryopreservedimmediately, using the protocols discussed herein below. Alternatively,the TIL population obtained from the first expansion, referred to as thesecond TIL population, can be subjected to a second expansion (which caninclude expansions sometimes referred to as REP) and then cryopreservedas discussed below. Similarly, in the case where genetically modifiedTILs will be used in therapy, the first TIL population (sometimesreferred to as the bulk TIL population) or the second TIL population(which can in some embodiments include populations referred to as theREP TIL populations) can be subjected to genetic modifications forsuitable treatments prior to expansion or after the first expansion andprior to the second expansion.

In some embodiments, the TILs obtained from the first expansion (forexample, from Step B as indicated in FIG. 9) are stored until phenotypedfor selection. In some embodiments, the TILs obtained from the firstexpansion (for example, from Step B as indicated in FIG. 9) are notstored and proceed directly to the second expansion. In someembodiments, the TILs obtained from the first expansion are notcryopreserved after the first expansion and prior to the secondexpansion. In some embodiments, the transition from the first expansionto the second expansion occurs at about 3 days, 4, days, 5 days, 6 days,7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 daysfrom when fragmentation occurs. In some embodiments, the transition fromthe first expansion to the second expansion occurs at about 3 days to 14days from when fragmentation occurs. In some embodiments, the transitionfrom the first expansion to the second expansion occurs at about 4 daysto 14 days from when fragmentation occurs. In some embodiments, thetransition from the first expansion to the second expansion occurs atabout 4 days to 10 days from when fragmentation occurs. In someembodiments, the transition from the first expansion to the secondexpansion occurs at about 7 days to 14 days from when fragmentationoccurs. In some embodiments, the transition from the first expansion tothe second expansion occurs at about 14 days from when fragmentationoccurs.

In some embodiments, the transition from the first expansion to thesecond expansion occurs at 1 day, 2 days, 3 days, 4 days, 5 days, 6days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14days from when fragmentation occurs. In some embodiments, the transitionfrom the first expansion to the second expansion occurs 1 day to 14 daysfrom when fragmentation occurs. In some embodiments, the first TILexpansion can proceed for 2 days to 14 days. In some embodiments, thetransition from the first expansion to the second expansion occurs 3days to 14 days from when fragmentation occurs. In some embodiments, thetransition from the first expansion to the second expansion occurs 4days to 14 days from when fragmentation occurs. In some embodiments, thetransition from the first expansion to the second expansion occurs 5days to 14 days from when fragmentation occurs.

In some embodiments, the transition from the first expansion to thesecond expansion occurs 6 days to 14 days from when fragmentationoccurs. In some embodiments, the transition from the first expansion tothe second expansion occurs 7 days to 14 days from when fragmentationoccurs. In some embodiments, the transition from the first expansion tothe second expansion occurs 8 days to 14 days from when fragmentationoccurs. In some embodiments, the transition from the first expansion tothe second expansion occurs 9 days to 14 days from when fragmentationoccurs. In some embodiments, the transition from the first expansion tothe second expansion occurs 10 days to 14 days from when fragmentationoccurs. In some embodiments, the transition from the first expansion tothe second expansion occurs 11 days to 14 days from when fragmentationoccurs. In some embodiments, the transition from the first expansion tothe second expansion occurs 12 days to 14 days from when fragmentationoccurs. In some embodiments, the transition from the first expansion tothe second expansion occurs 13 days to 14 days from when fragmentationoccurs. In some embodiments, the transition from the first expansion tothe second expansion occurs 14 days from when fragmentation occurs. Insome embodiments, the transition from the first expansion to the secondexpansion occurs 1 day to 11 days from when fragmentation occurs. Insome embodiments, the transition from the first expansion to the secondexpansion occurs 2 days to 11 days from when fragmentation occurs. Insome embodiments, the transition from the first expansion to the secondexpansion occurs 3 days to 11 days from when fragmentation occurs. Insome embodiments, the transition from the first expansion to the secondexpansion occurs 4 days to 11 days from when fragmentation occurs. Insome embodiments, the transition from the first expansion to the secondexpansion occurs 5 days to 11 days from when fragmentation occurs. Insome embodiments, the transition from the first expansion to the secondexpansion occurs 6 days to 11 days from when fragmentation occurs. Insome embodiments, the transition from the first expansion to the secondexpansion occurs 7 days to 11 days from when fragmentation occurs. Insome embodiments, the transition from the first expansion to the secondexpansion occurs 8 days to 11 days from when fragmentation occurs. Insome embodiments, the transition from the first expansion to the secondexpansion occurs 9 days to 11 days from when fragmentation occurs. Insome embodiments, the transition from the first expansion to the secondexpansion occurs 10 days to 11 days from when fragmentation occurs. Insome embodiments, the transition from the first expansion to the secondexpansion occurs 11 days from when fragmentation occurs.

In some embodiments, the TILs are not stored after the first expansionand prior to the second expansion, and the TILs proceed directly to thesecond expansion (for example, in some embodiments, there is no storageduring the transition from Step B to Step D as shown in FIG. 9). In someembodiments, the transition occurs in closed system, as describedherein. In some embodiments, the TILs from the first expansion, thesecond population of TILs, proceeds directly into the second expansionwith no transition period.

In some embodiments, the transition from the first expansion to thesecond expansion, for example, Step C according to FIG. 9, is performedin a closed system bioreactor. In some embodiments, a closed system isemployed for the TIL expansion, as described herein. In someembodiments, a single bioreactor is employed. In some embodiments, thesingle bioreactor employed is for example a G-REX-10 or a G-REX-100. Insome embodiments, the closed system bioreactor is a single bioreactor.

D. STEP D: Second Expansion

In some embodiments, the TIL cell population is expanded in number afterharvest and initial bulk processing for example, after Step A and StepB, and the transition referred to as Step C, as indicated in FIG. 9).This further expansion is referred to herein as the second expansion,which can include expansion processes generally referred to in the artas a rapid expansion process (REP; as well as processes as indicated inStep D of FIG. 9). The second expansion is generally accomplished usinga culture media comprising a number of components, including feedercells, a cytokine source, and an anti-CD3 antibody, in a gas-permeablecontainer.

In some embodiments, the second expansion or second TIL expansion (whichcan include expansions sometimes referred to as REP; as well asprocesses as indicated in Step D of FIG. 9) of TIL can be performedusing any TIL flasks or containers known by those of skill in the art.In some embodiments, the second TIL expansion can proceed for 7 days, 8days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In someembodiments, the second TIL expansion can proceed for about 7 days toabout 14 days. In some embodiments, the second TIL expansion can proceedfor about 8 days to about 14 days. In some embodiments, the second TILexpansion can proceed for about 9 days to about 14 days. In someembodiments, the second TIL expansion can proceed for about 10 days toabout 14 days. In some embodiments, the second TIL expansion can proceedfor about 11 days to about 14 days. In some embodiments, the second TILexpansion can proceed for about 12 days to about 14 days. In someembodiments, the second TIL expansion can proceed for about 13 days toabout 14 days. In some embodiments, the second TIL expansion can proceedfor about 14 days.

In an embodiment, the second expansion can be performed in a gaspermeable container using the methods of the present disclosure(including for example, expansions referred to as REP; as well asprocesses as indicated in Step D of FIG. 9). For example, TILs can berapidly expanded using non-specific T-cell receptor stimulation in thepresence of interleukin-2 (IL-2) or interleukin-15 (IL-15). Thenon-specific T-cell receptor stimulus can include, for example, ananti-CD3 antibody, such as about 30 ng/ml of OKT3, a mouse monoclonalanti-CD3 antibody (commercially available from Ortho-McNeil, Raritan,N.J. or Miltenyi Biotech, Auburn, Calif.) or UHCT-1 (commerciallyavailable from BioLegend, San Diego, Calif., USA). TILs can be expandedto induce further stimulation of the TILs in vitro by including one ormore antigens during the second expansion, including antigenic portionsthereof, such as epitope(s), of the cancer, which can be optionallyexpressed from a vector, such as a human leukocyte antigen A2 (HLA-A2)binding peptide, e.g., 0.3 μM MART-1:26-35 (27 L) or gpl 00:209-217(210M), optionally in the presence of a T-cell growth factor, such as300 IU/mL IL-2 or IL-15. Other suitable antigens may include, e.g.,NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, andVEGFR2, or antigenic portions thereof. TIL may also be rapidly expandedby re-stimulation with the same antigen(s) of the cancer pulsed ontoHLA-A2-expressing antigen-presenting cells. Alternatively, the TILs canbe further re-stimulated with, e.g., example, irradiated, autologouslymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2.In some embodiments, the re-stimulation occurs as part of the secondexpansion. In some embodiments, the second expansion occurs in thepresence of irradiated, autologous lymphocytes or with irradiatedHLA-A2+ allogeneic lymphocytes and IL-2.

In an embodiment, the cell culture medium further comprises IL-2. In asome embodiments, the cell culture medium comprises about 3000 IU/mL ofIL-2. In an embodiment, the cell culture medium comprises about 1000IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000IU/mL, about 3500 IU/mL, about 4000 IU/mL, about 4500 IU/mL, about 5000IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000IU/mL, about 7500 IU/mL, or about 8000 IU/mL of IL-2. In an embodiment,the cell culture medium comprises between 1000 and 2000 IU/mL, between2000 and 3000 IU/mL, between 3000 and 4000 IU/mL, between 4000 and 5000IU/mL, between 5000 and 6000 IU/mL, between 6000 and 7000 IU/mL, between7000 and 8000 IU/mL, or between 8000 IU/mL of IL-2.

In an embodiment, the cell culture medium comprises OKT3 antibody. TheOKT-3 antibody may be present in the cell culture medium beginning onday 0 of the REP (i.e., the start day of the REP) and/or day 0 of thesecond expansion (i.e., the start day of the second expansion). In someembodiments, the cell culture medium comprises about 30 ng/mL of OKT3antibody. In an embodiment, the cell culture medium comprises about 0.1ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about 5 ng/mL,about 7.5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL,about 100 ng/mL, about 200 ng/mL, about 500 ng/mL, and about 1 μg/mL ofOKT3 antibody. In an embodiment, the cell culture medium comprisesbetween 0.1 ng/mL and 1 ng/mL, between 1 ng/mL and 5 ng/mL, between 5ng/mL and 10 ng/mL, between 10 ng/mL and 20 ng/mL, between 20 ng/mL and30 ng/mL, between 30 ng/mL and 40 ng/mL, between 40 ng/mL and 50 ng/mL,and between 50 ng/mL and 100 ng/mL of OKT3 antibody. In someembodiments, the cell culture medium does not comprise OKT-3 antibody.

In some embodiments, a combination of IL-2, IL-7, IL-15, and/or IL-21are employed as a combination during the second expansion. In someembodiments, IL-2, IL-7, IL-15, and/or IL-21 as well as any combinationsthereof can be included during the second expansion, including forexample during a Step D processes according to FIG. 9, as well asdescribed herein. In some embodiments, a combination of IL-2, IL-15, andIL-21 are employed as a combination during the second expansion. In someembodiments, IL-2, IL-15, and IL-21 as well as any combinations thereofcan be included during Step D processes according to FIG. 9 and asdescribed herein.

In some embodiments, the second expansion can be conducted in asupplemented cell culture medium comprising IL-2, OKT-3, andantigen-presenting feeder cells. In some embodiments, the secondexpansion occurs in a supplemented cell culture medium. In someembodiments, the supplemented cell culture medium comprises IL-2, OKT-3,and antigen-presenting feeder cells. In some embodiments, the secondcell culture medium comprises IL-2, OKT-3, and antigen-presenting cells(APCs; also referred to as antigen-presenting feeder cells). In someembodiments, the second expansion occurs in a cell culture mediumcomprising IL-2, OKT-3, and antigen-presenting feeder cells (i.e.,antigen presenting cells).

In some embodiments, the second expansion culture media comprises about500 IU/mL of IL-15, about 400 IU/mL of IL-15, about 300 IU/mL of IL-15,about 200 IU/mL of IL-15, about 180 IU/mL of IL-15, about 160 IU/mL ofIL-15, about 140 IU/mL of IL-15, about 120 IU/mL of IL-15, or about 100IU/mL of IL-15. In some embodiments, the second expansion culture mediacomprises about 500 IU/mL of IL-15 to about 100 IU/mL of IL-15. In someembodiments, the second expansion culture media comprises about 400IU/mL of IL-15 to about 100 IU/mL of IL-15. In some embodiments, thesecond expansion culture media comprises about 300 IU/mL of IL-15 toabout 100 IU/mL of IL-15. In some embodiments, the second expansionculture media comprises about 200 IU/mL of IL-15. In some embodiments,the cell culture medium comprises about 180 IU/mL of IL-15. In anembodiment, the cell culture medium further comprises IL-15. In apreferred embodiment, the cell culture medium comprises about 180 IU/mLof IL-15.

In some embodiments, the second expansion culture media comprises about20 IU/mL of IL-21, about 15 IU/mL of IL-21, about 12 IU/mL of IL-21,about 10 IU/mL of IL-21, about 5 IU/mL of IL-21, about 4 IU/mL of IL-21,about 3 IU/mL of IL-21, about 2 IU/mL of IL-21, about 1 IU/mL of IL-21,or about 0.5 IU/mL of IL-21. In some embodiments, the second expansionculture media comprises about 20 IU/mL of IL-21 to about 0.5 IU/mL ofIL-21. In some embodiments, the second expansion culture media comprisesabout 15 IU/mL of IL-21 to about 0.5 IU/mL of IL-21. In someembodiments, the second expansion culture media comprises about 12 IU/mLof IL-21 to about 0.5 IU/mL of IL-21. In some embodiments, the secondexpansion culture media comprises about 10 IU/mL of IL-21 to about 0.5IU/mL of IL-21. In some embodiments, the second expansion culture mediacomprises about 5 IU/mL of IL-21 to about 1 IU/mL of IL-21. In someembodiments, the second expansion culture media comprises about 2 IU/mLof IL-21. In some embodiments, the cell culture medium comprises about 1IU/mL of IL-21. In some embodiments, the cell culture medium comprisesabout 0.5 IU/mL of IL-21. In an embodiment, the cell culture mediumfurther comprises IL-21. In a preferred embodiment, the cell culturemedium comprises about 1 IU/mL of IL-21.

In some embodiments the antigen-presenting feeder cells (APCs) arePBMCs. In an embodiment, the ratio of TILs to PBMCs and/orantigen-presenting cells in the rapid expansion and/or the secondexpansion is about 1 to 25, about 1 to 50, about 1 to 100, about 1 to125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225,about 1 to 250, about 1 to 275, about 1 to 300, about 1 to 325, about 1to 350, about 1 to 375, about 1 to 400, or about 1 to 500. In anembodiment, the ratio of TILs to PBMCs in the rapid expansion and/or thesecond expansion is between 1 to 50 and 1 to 300. In an embodiment, theratio of TILs to PBMCs in the rapid expansion and/or the secondexpansion is between 1 to 100 and 1 to 200.

In an embodiment, REP and/or the second expansion is performed in flaskswith the bulk TILs being mixed with a 100- or 200-fold excess ofinactivated feeder cells, 30 mg/mL OKT3 anti-CD3 antibody and 3000 IU/mLIL-2 in 150 ml media. Media replacement is done (generally 2/3 mediareplacement via respiration with fresh media) until the cells aretransferred to an alternative growth chamber. Alternative growthchambers include G-REX flasks and gas permeable containers as more fullydiscussed below.

In some embodiments, the second expansion (which can include processesreferred to as the REP process) is shortened to 7-14 days, as discussedin the examples and figures. In some embodiments, the second expansionis shortened to 11 days.

In an embodiment, REP and/or the second expansion may be performed usingT-175 flasks and gas permeable bags as previously described (Tran, etal., J Immunother. 2008, 31, 742-51; Dudley, et al., J Immunother. 2003,26, 332-42) or gas permeable cultureware (G-Rex flasks). In someembodiments, the second expansion (including expansions referred to asrapid expansions) is performed in T-175 flasks, and about 1×10⁶ TILssuspended in 150 mL of media may be added to each T-175 flask. The TILsmay be cultured in a 1 to 1 mixture of CM and AIM-V medium, supplementedwith 3000 IU per mL of IL-2 and 30 ng per ml of anti-CD3. The T-175flasks may be incubated at 37° C. in 5% CO₂. Half the media may beexchanged on day 5 using 50/50 medium with 3000 IU per mL of IL-2. Insome embodiments, on day 7 cells from two T-175 flasks may be combinedin a 3 L bag and 300 mL of AIM V with 5% human AB serum and 3000 IU permL of IL-2 was added to the 300 ml of TIL suspension. The number ofcells in each bag was counted every day or two and fresh media was addedto keep the cell count between 0.5 and 2.0×10⁶ cells/mL.

In an embodiment, the second expansion (which can include expansionsreferred to as REP, as well as those referred to in Step D of FIG. 9)may be performed in 500 mL capacity gas permeable flasks with 100 cmgas-permeable silicon bottoms (G-Rex 100, commercially available fromWilson Wolf Manufacturing Corporation, New Brighton, Minn., USA), 5×10⁶or 10×10⁶ TIL may be cultured with PBMCs in 400 mL of 50/50 medium,supplemented with 5% human AB serum, 3000 IU per mL of IL-2 and 30 ngper ml of anti-CD3 (OKT3). The G-Rex 100 flasks may be incubated at 37°C. in 5% CO₂. On day 5, 250 mL of supernatant may be removed and placedinto centrifuge bottles and centrifuged at 1500 rpm (491×g) for 10minutes. The TIL pellets may be re-suspended with 150 mL of fresh mediumwith 5% human AB serum, 3000 IU per mL of IL-2, and added back to theoriginal G-Rex 100 flasks. When TIL are expanded serially in G-Rex 100flasks, on day 7 the TIL in each G-Rex 100 may be suspended in the 300mL of media present in each flask and the cell suspension may be dividedinto 3 100 mL aliquots that may be used to seed 3 G-Rex 100 flasks. Then150 mL of AIM-V with 5% human AB serum and 3000 IU per mL of IL-2 may beadded to each flask. The G-Rex 100 flasks may be incubated at 37° C. in5% CO₂ and after 4 days 150 mL of AIM-V with 3000 IU per mL of IL-2 maybe added to each G-REX 100 flask. The cells may be harvested on day 14of culture.

In an embodiment, the second expansion (including expansions referred toas REP) is performed in flasks with the bulk TILs being mixed with a100- or 200-fold excess of inactivated feeder cells, 30 mg/mL OKT3anti-CD3 antibody and 3000 IU/mL IL-2 in 150 ml media. In someembodiments, media replacement is done until the cells are transferredto an alternative growth chamber. In some embodiments, 2/3 of the mediais replaced by respiration with fresh media. In some embodiments,alternative growth chambers include G-REX flasks and gas permeablecontainers as more fully discussed below.

In an embodiment, the second expansion (including expansions referred toas REP) is performed and further comprises a step wherein TILs areselected for superior tumor reactivity. Any selection method known inthe art may be used. For example, the methods described in U.S. PatentApplication Publication No. 2016/0010058 A1, the disclosures of whichare incorporated herein by reference, may be used for selection of TILsfor superior tumor reactivity.

Optionally, a cell viability assay can be performed after the secondexpansion (including expansions referred to as the REP expansion), usingstandard assays known in the art. For example, a trypan blue exclusionassay can be done on a sample of the bulk TILs, which selectively labelsdead cells and allows a viability assessment. In some embodiments, TILsamples can be counted and viability determined using a Cellometer K2automated cell counter (Nexcelom Bioscience, Lawrence, Mass.). In someembodiments, viability is determined according to the Cellometer K2Image Cytometer Automatic Cell Counter protocol described, for example,in Example 15.

In some embodiments, the second expansion (including expansions referredto as REP) of TIL can be performed using T-175 flasks and gas-permeablebags as previously described (Tran K Q, Zhou J, Durflinger K H, et al.,2008, J Immunother., 31:742-751, and Dudley M E, Wunderlich J R, SheltonT E, et al. 2003, J Immunother., 26:332-342) or gas-per-meable G-Rexflasks. In some embodiments, the second expansion is performed usingflasks. In some embodiments, the second expansion is performed usinggas-permeable G-Rex flasks. In some embodiments, the second expansion isperformed in T-175 flasks, and about 1×10⁶ TIL are suspended in about150 mL of media and this is added to each T-175 flask. The TIL arecultured with irradiated (50 Gy) allogeneic PBMC as “feeder” cells at aratio of 1 to 100 and the cells were cultured in a 1 to 1 mixture of CMand AIM-V medium (50/50 medium), supplemented with 3000 IU/mL of IL-2and 30 ng/mL of anti-CD3. The T-175 flasks are incubated at 37° C. in 5%CO₂. In some embodiments, half the media is changed on day 5 using 50/50medium with 3000 IU/mL of IL-2. In some embodiments, on day 7, cellsfrom 2 T-175 flasks are combined in a 3 L bag and 300 mL of AIM-V with5% human AB serum and 3000 IU/mL of IL-2 is added to the 300 mL of TILsuspension. The number of cells in each bag can be counted every day ortwo and fresh media can be added to keep the cell count between about0.5 and about 2.0×10⁶ cells/mL.

In some embodiments, the second expansion (including expansions referredto as REP) are performed in 500 mL capacity flasks with 100 cm²gas-permeable silicon bottoms (G-Rex 100, Wilson Wolf) (FIG. 1), about5×10⁶ or 10×10⁶ TIL are cultured with irradiated allogeneic PBMC at aratio of 1 to 100 in 400 mL of 50/50 medium, supplemented with 3000IU/mL of IL-2 and 30 ng/mL of anti-CD3. The G-Rex 100 flasks areincubated at 37° C. in 5% CO₂. In some embodiments, on day 5, 250 mL ofsupernatant is removed and placed into centrifuge bottles andcentrifuged at 1500 rpm (491 g) for 10 minutes. The TIL pellets can thenbe resuspended with 150 mL of fresh 50/50 medium with 3000 IU/mL of IL-2and added back to the original G-Rex 100 flasks. In embodiments whereTILs are expanded serially in G-Rex 100 flasks, on day 7 the TIL in eachG-Rex 100 are suspended in the 300 mL of media present in each flask andthe cell suspension was divided into three 100 mL aliquots that are usedto seed 3 G-Rex 100 flasks. Then 150 mL of AIM-V with 5% human AB serumand 3000 IU/mL of IL-2 is added to each flask. The G-Rex 100 flasks areincubated at 37° C. in 5% CO₂ and after 4 days 150 mL of AIM-V with 3000IU/mL of IL-2 is added to each G-Rex 100 flask. The cells are harvestedon day 14 of culture.

The diverse antigen receptors of T and B lymphocytes are produced bysomatic recombination of a limited, but large number of gene segments.These gene segments: V (variable), D (diversity), J (joining), and C(constant), determine the binding specificity and downstreamapplications of immunoglobulins and T-cell receptors (TCRs). The presentinvention provides a method for generating TILs which exhibit andincrease the T-cell repertoire diversity. In some embodiments, the TILsobtained by the present method exhibit an increase in the T-cellrepertoire diversity. In some embodiments, the TILs obtained in thesecond expansion exhibit an increase in the T-cell repertoire diversity.In some embodiments, the increase in diversity is an increase in theimmunoglobulin diversity and/or the T-cell receptor diversity. In someembodiments, the diversity is in the immunoglobulin is in theimmunoglobulin heavy chain. In some embodiments, the diversity is in theimmunoglobulin is in the immunoglobulin light chain. In someembodiments, the diversity is in the T-cell receptor. In someembodiments, the diversity is in one of the T-cell receptors selectedfrom the group consisting of alpha, beta, gamma, and delta receptors. Insome embodiments, there is an increase in the expression of T-cellreceptor (TCR) alpha and/or beta. In some embodiments, there is anincrease in the expression of T-cell receptor (TCR) alpha. In someembodiments, there is an increase in the expression of T-cell receptor(TCR) beta. In some embodiments, there is an increase in the expressionof TCRab (i.e., TCRα/β).

In some embodiments, the second expansion culture medium (e.g.,sometimes referred to as CM2 or the second cell culture medium),comprises IL-2, OKT-3, as well as the antigen-presenting feeder cells(APCs), as discussed in more detail below.

In some embodiments, the second expansion, for example, Step D accordingto FIG. 9, is performed in a closed system bioreactor. In someembodiments, a closed system is employed for the TIL expansion, asdescribed herein. In some embodiments, a single bioreactor is employed.In some embodiments, the single bioreactor employed is for example aG-REX-10 or a G-REX-100. In some embodiments, the closed systembioreactor is a single bioreactor.

1. Feeder Cells and Antigen Presenting Cells

In an embodiment, the second expansion procedures described herein (forexample including expansion such as those described in Step D from FIG.9, as well as those referred to as REP) require an excess of feedercells during REP TIL expansion and/or during the second expansion. Inmany embodiments, the feeder cells are peripheral blood mononuclearcells (PBMCs) obtained from standard whole blood units from healthyblood donors. The PBMCs are obtained using standard methods such asFicoll-Paque gradient separation.

In general, the allogenic PBMCs are inactivated, either via irradiationor heat treatment, and used in the REP procedures, as described in theexamples, in particular example 14, which provides an exemplary protocolfor evaluating the replication incompetence of irradiate allogeneicPBMCs.

In some embodiments, PBMCs are considered replication incompetent andaccepted for use in the TIL expansion procedures described herein if thetotal number of viable cells on day 14 is less than the initial viablecell number put into culture on day 0 of the REP and/or day 0 of thesecond expansion (i.e., the start day of the second expansion). See, forexample, Example 14.

In some embodiments, PBMCs are considered replication incompetent andaccepted for use in the TIL expansion procedures described herein if thetotal number of viable cells, cultured in the presence of OKT3 and IL-2,on day 7 and day 14 has not increased from the initial viable cellnumber put into culture on day 0 of the REP and/or day 0 of the secondexpansion (i.e., the start day of the second expansion). In someembodiments, the PBMCs are cultured in the presence of 30 ng/ml OKT3antibody and 3000 IU/ml IL-2. See, for example, Example 13.

In some embodiments, PBMCs are considered replication incompetent andaccepted for use in the TIL expansion procedures described herein if thetotal number of viable cells, cultured in the presence of OKT3 and IL-2,on day 7 and day 14 has not increased from the initial viable cellnumber put into culture on day 0 of the REP and/or day 0 of the secondexpansion (i.e., the start day of the second expansion). In someembodiments, the PBMCs are cultured in the presence of 5-60 ng/ml OKT3antibody and 1000-6000 IU/ml IL-2. In some embodiments, the PBMCs arecultured in the presence of 10-50 ng/ml OKT3 antibody and 2000-5000IU/ml IL-2. In some embodiments, the PBMCs are cultured in the presenceof 20-40 ng/ml OKT3 antibody and 2000-4000 IU/ml IL-2. In someembodiments, the PBMCs are cultured in the presence of 25-35 ng/ml OKT3antibody and 2500-3500 IU/ml IL-2.

In some embodiments, the antigen-presenting feeder cells are PBMCs. Insome embodiments, the antigen-presenting feeder cells are artificialantigen-presenting feeder cells. In an embodiment, the ratio of TILs toantigen-presenting feeder cells in the second expansion is about 1 to25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375,about 1 to 400, or about 1 to 500. In an embodiment, the ratio of TILsto antigen-presenting feeder cells in the second expansion is between 1to 50 and 1 to 300. In an embodiment, the ratio of TILs toantigen-presenting feeder cells in the second expansion is between 1 to100 and 1 to 200.

In an embodiment, the second expansion procedures described hereinrequire a ratio of about 2.5×10⁹ feeder cells to about 100×10⁶ TILs. Inanother embodiment, the second expansion procedures described hereinrequire a ratio of about 2.5×10⁹ feeder cells to about 50×10⁶ TILs. Inyet another embodiment, the second expansion procedures described hereinrequire about 2.5×10⁹ feeder cells to about 25×10⁶ TILs.

In an embodiment, the second expansion procedures described hereinrequire an excess of feeder cells during the second expansion. In manyembodiments, the feeder cells are peripheral blood mononuclear cells(PBMCs) obtained from standard whole blood units from healthy blooddonors. The PBMCs are obtained using standard methods such asFicoll-Paque gradient separation. In an embodiment, artificialantigen-presenting (aAPC) cells are used in place of PBMCs.

In general, the allogenic PBMCs are inactivated, either via irradiationor heat treatment, and used in the TIL expansion procedures describedherein, including the exemplary procedures described in FIGS. 4, 5, and9.

In an embodiment, artificial antigen presenting cells are used in thesecond expansion as a replacement for, or in combination with, PBMCs.

2. Cytokines

The expansion methods described herein generally use culture media withhigh doses of a cytokine, in particular IL-2, as is known in the art.

Alternatively, using combinations of cytokines for the rapid expansionand or second expansion of TILS is additionally possible, withcombinations of two or more of IL-2, IL-15 and IL-21 as is generallyoutlined in International Publication No. WO 2015/189356 and WInternational Publication No. WO 2015/189357, hereby expresslyincorporated by reference in their entirety. Thus, possible combinationsinclude IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21 and IL-2, IL-15and IL-21, with the latter finding particular use in many embodiments.The use of combinations of cytokines specifically favors the generationof lymphocytes, and in particular T-cells as described therein. 3.Anti-CD3 Antibodies

In some embodiments, the culture media used in expansion methodsdescribed herein (including those referred to as REP, see for example,FIG. 9) also includes an anti-CD3 antibody. An anti-CD3 antibody incombination with IL-2 induces T cell activation and cell division in theTIL population. This effect can be seen with full length antibodies aswell as Fab and F(ab′)2 fragments, with the former being generallypreferred; see, e.g., Tsoukas et al., J. Immunol. 1985, 135, 1719,hereby incorporated by reference in its entirety.

As will be appreciated by those in the art, there are a number ofsuitable anti-human CD3 antibodies that find use in the invention,including anti-human CD3 polyclonal and monoclonal antibodies fromvarious mammals, including, but not limited to, murine, human, primate,rat, and canine antibodies. In particular embodiments, the OKT3 anti-CD3antibody is used (commercially available from Ortho-McNeil, Raritan,N.J. or Miltenyi Biotech, Auburn, Calif.).

In some embodiments, the anti-CD3 antibody, such as OKT3, may be addedto TIL culture 2 days, 3 days, 4 days, or 5 days prior to anelectroporation step. In some embodiments, the anti-CD3 antibody, suchas OKT3, may be added immediately before an electroporation step. Insome embodiments, the anti-CD3 antibody, such as OKT3, may be addedimmediately after an electroporation step. In some embodiments, theanti-CD3 antibody, such as OKT3, may be added 2 days, 3 days, 4 days, or5 days after an electroporation step.

4. 4-1BB and OX40 Agonists

According to an embodiment, the cell culture medium further comprises a4-1BB (CD137) agonist and/or an OX40 agonist during the first expansion,the second expansion, or both. The gene-editing may be carried out afterthe 4-1BB agonist and/or the OX40 agonist are introduced into the cellculture medium. Alternatively, the gene-editing may be carried outbefore the 4-1BB agonist and/or the OX40 agonist are introduced into thecell culture medium.

The 4-1BB agonist may be any 4-1BB binding molecule known in the art.The 4-1BB binding molecule may be a monoclonal antibody or fusionprotein capable of binding to human or mammalian 4-1BB. The 4-1BBagonists or 4-1BB binding molecules may comprise an immunoglobulin heavychain of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class(e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass ofimmunoglobulin molecule. The 4-1BB agonist or 4-1BB binding molecule mayhave both a heavy and a light chain. As used herein, the term bindingmolecule also includes antibodies (including full length antibodies),monoclonal antibodies (including full length monoclonal antibodies),polyclonal antibodies, multispecific antibodies (e.g., bispecificantibodies), human, humanized or chimeric antibodies, and antibodyfragments, e.g., Fab fragments, F(ab′) fragments, fragments produced bya Fab expression library, epitope-binding fragments of any of the above,and engineered forms of antibodies, e.g., scFv molecules, that bind to4-1BB. In an embodiment, the 4-1BB agonist is an antigen binding proteinthat is a fully human antibody. In an embodiment, the 4-1BB agonist isan antigen binding protein that is a humanized antibody. In someembodiments, 4-1BB agonists for use in the presently disclosed methodsand compositions include anti-4-1BB antibodies, human anti-4-1BBantibodies, mouse anti-4-1BB antibodies, mammalian anti-4-1BBantibodies, monoclonal anti-4-1BB antibodies, polyclonal anti-4-1BBantibodies, chimeric anti-4-1BB antibodies, anti-4-1BB adnectins,anti-4-1BB domain antibodies, single chain anti-4-1BB fragments, heavychain anti-4-1BB fragments, light chain anti-4-1BB fragments, anti-4-1BBfusion proteins, and fragments, derivatives, conjugates, variants, orbiosimilars thereof. Agonistic anti-4-1BB antibodies are known to inducestrong immune responses. Lee, et al., PLOS One 2013, 8, e69677. In apreferred embodiment, the 4-1BB agonist is an agonistic, anti-4-1BBhumanized or fully human monoclonal antibody (i.e., an antibody derivedfrom a single cell line). In an embodiment, the 4-1BB agonist is EU-101(Eutilex Co. Ltd.), utomilumab, or urelumab, or a fragment, derivative,conjugate, variant, or biosimilar thereof. In a preferred embodiment,the 4-1BB agonist is utomilumab or urelumab, or a fragment, derivative,conjugate, variant, or biosimilar thereof.

In a preferred embodiment, the 4-1BB agonist or 4-1BB binding moleculemay also be a fusion protein. In a preferred embodiment, a multimeric4-1BB agonist, such as a trimeric or hexameric 4-1BB agonist (with threeor six ligand binding domains), may induce superior receptor (4-1BBL)clustering and internal cellular signaling complex formation compared toan agonistic monoclonal antibody, which typically possesses two ligandbinding domains. Trimeric (trivalent) or hexameric (or hexavalent) orgreater fusion proteins comprising three TNFRSF binding domains andIgG1-Fc and optionally further linking two or more of these fusionproteins are described, e.g., in Gieffers, et al., Mol. CancerTherapeutics 2013, 12, 2735-47.

Agonistic 4-1BB antibodies and fusion proteins are known to inducestrong immune responses. In a preferred embodiment, the 4-1BB agonist isa monoclonal antibody or fusion protein that binds specifically to 4-1BBantigen in a manner sufficient to reduce toxicity. In some embodiments,the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusionprotein that abrogates antibody-dependent cellular toxicity (ADCC), forexample NK cell cytotoxicity. In some embodiments, the 4-1BB agonist isan agonistic 4-1BB monoclonal antibody or fusion protein that abrogatesantibody-dependent cell phagocytosis (ADCP). In some embodiments, the4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusionprotein that abrogates complement-dependent cytotoxicity (CDC). In someembodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibodyor fusion protein which abrogates Fc region functionality.

In some embodiments, the 4-1BB agonists are characterized by binding tohuman 4-1BB (SEQ ID NO:9) with high affinity and agonistic activity. Inan embodiment, the 4-1BB agonist is a binding molecule that binds tohuman 4-1BB (SEQ ID NO:9). In an embodiment, the 4-1BB agonist is abinding molecule that binds to murine 4-1BB (SEQ ID NO:10). The aminoacid sequences of 4-1BB antigen to which a 4-1BB agonist or bindingmolecule binds are summarized in Table 3.

TABLE 3 Amino acid sequences of 4-1BB antigens. IdentifierSequence (One-Letter Amino Acid Symbols) SEQ ID NO: 9MGNSCYNIVA TLLLVLNFER TRSLQDPCSN CPAGTFCDNN RNQICSPCPP NSFSSAGGQR  60human 4-1BB,TCDICRQCKG VFRTRKECSS TSNAECDCTP GFHCLGAGCS MCEQDCKQGQ ELTKKGCKDC 120Tumor necrosisCFGTFNDQKR GICRPWTNCS LDGKSVLVNG TKERDVVCGP SPADLSPGAS SVTPPAPARE 180factor receptorPGHSPQIISF FLALTSTALL FLLFFLTLRF SVVKRGRKKL LYIFKQPFMR PVQTTQEEDG 240superfamily,CSCRFPEEEE GGCEL                                                  255member 9 (Homo sapiens) SEQ ID NO: 10MGNNCYNVVV IVLLLVGCEK VGAVQNSCDN CQPGTFCRKY NPVCKSCPPS TFSSIGGQPN  60murine 4-1BB,CNICRVCAGY FRFKKFCSST HNAECECIEG FHCLGPQCTR CEKDCRPGQE LTKQGCKTCS 120Tumor necrosisLGTFNDQNGT GVCRPWTNCS LDGRSVLKTG TTEKDVVCGP PVVSFSPSTT ISVTPEGGPG 180factor receptorGHSLQVLTLF LALTSALLLA LIFITLLFSV LKWIRKKFPH IFKQPFKKTT GAAQEEDACS 240superfamily,CRCPQEEEGG GGGYEL                                                 256member 9 (Mus musculus)

In some embodiments, the compositions, processes and methods describedinclude a 4-1BB agonist that binds human or murine 4-1BB with a K_(D) ofabout 100 pM or lower, binds human or murine 4-1BB with a K_(D) of about90 pM or lower, binds human or murine 4-1BB with a K_(D) of about 80 pMor lower, binds human or murine 4-1BB with a K_(D) of about 70 pM orlower, binds human or murine 4-1BB with a K_(D) of about 60 pM or lower,binds human or murine 4-1BB with a K_(D) of about 50 pM or lower, bindshuman or murine 4-1BB with a K_(D) of about 40 pM or lower, or bindshuman or murine 4-1BB with a K_(D) of about 30 pM or lower.

In some embodiments, the compositions, processes and methods describedinclude a 4-1BB agonist that binds to human or murine 4-1BB with ak_(assoc) of about 7.5×10⁵ l/M·s or faster, binds to human or murine4-1BB with a k_(assoc) of about 7.5×10⁵ l/M·s or faster, binds to humanor murine 4-1BB with a k_(assoc) of about 8×10⁵ l/M·s or faster, bindsto human or murine 4-1BB with a k_(assoc) of about 8.5×10⁵ l/M·s orfaster, binds to human or murine 4-1BB with a k_(assoc) of about 9×10⁵l/M·s or faster, binds to human or murine 4-1BB with a k_(assoc) ofabout 9.5×10⁵ l/M·s or faster, or binds to human or murine 4-1BB with ak_(assoc) of about 1×10⁶ l/M·s or faster.

In some embodiments, the compositions, processes and methods describedinclude a 4-1BB agonist that binds to human or murine 4-1BB with ak_(dissoc) of about 2×10⁻⁵ l/s or slower, binds to human or murine 4-1BBwith a k_(dissoc) of about 2.1×10⁻⁵ l/s or slower, binds to human ormurine 4-1BB with a k_(dissoc) of about 2.2×10⁻⁵ l/s or slower, binds tohuman or murine 4-1BB with a k_(dissoc) of about 2.3×10⁻⁵ l/s or slower,binds to human or murine 4-1BB with a k_(dissoc) of about 2.4×10⁻⁵ l/sor slower, binds to human or murine 4-1BB with a k_(dissoc) of about2.5×10⁻⁵ l/s or slower, binds to human or murine 4-1BB with a k_(dissoc)of about 2.6×10⁻⁵ l/s or slower or binds to human or murine 4-1BB with ak_(dissoc) of about 2.7×10⁻⁵ l/s or slower, binds to human or murine4-1BB with a k_(dissoc) of about 2.8×10⁻⁵ l/s or slower, binds to humanor murine 4-1BB with a k_(dissoc) of about 2.9×10⁻⁵ l/s or slower, orbinds to human or murine 4-1BB with a k_(dissoc) of about 3×10⁻⁵ l/s orslower.

In some embodiments, the compositions, processes and methods describedinclude a 4-1BB agonist that binds to human or murine 4-1BB with an IC₅₀of about 10 nM or lower, binds to human or murine 4-1BB with an IC₅₀ ofabout 9 nM or lower, binds to human or murine 4-1BB with an IC₅₀ ofabout 8 nM or lower, binds to human or murine 4-1BB with an IC₅₀ ofabout 7 nM or lower, binds to human or murine 4-1BB with an IC₅₀ ofabout 6 nM or lower, binds to human or murine 4-1BB with an IC₅₀ ofabout 5 nM or lower, binds to human or murine 4-1BB with an IC₅₀ ofabout 4 nM or lower, binds to human or murine 4-1BB with an IC₅₀ ofabout 3 nM or lower, binds to human or murine 4-1BB with an IC₅₀ ofabout 2 nM or lower, or binds to human or murine 4-1BB with an IC₅₀ ofabout 1 nM or lower.

In a preferred embodiment, the 4-1BB agonist is utomilumab, also knownas PF-05082566 or MOR-7480, or a fragment, derivative, variant, orbiosimilar thereof. Utomilumab is available from Pfizer, Inc. Utomilumabis an immunoglobulin G2-lambda, anti-[Homo sapiens TNFRSF9 (tumornecrosis factor receptor (TNFR) superfamily member 9, 4-1BB, T cellantigen ILA, CD137)], Homo sapiens (fully human) monoclonal antibody.The amino acid sequences of utomilumab are set forth in Table 4.Utomilumab comprises glycosylation sites at Asn59 and Asn292; heavychain intrachain disulfide bridges at positions 22-96 (V_(H)-V_(L)),143-199 (C_(H)1-C_(L)), 256-316 (C_(H)2) and 362-420 (C_(H)3); lightchain intrachain disulfide bridges at positions 22′-87′ (V_(H)-V_(L))and 136′-195′ (C_(H)1-C_(L)); interchain heavy chain-heavy chaindisulfide bridges at IgG2A isoform positions 218-218, 219-219, 222-222,and 225-225, at IgG2A/B isoform positions 218-130, 219-219, 222-222, and225-225, and at IgG2B isoform positions 219-130 (2), 222-222, and225-225; and interchain heavy chain-light chain disulfide bridges atIgG2A isoform positions 130-213′ (2), IgG2A/B isoform positions 218-213′and 130-213′, and at IgG2B isoform positions 218-213′ (2). Thepreparation and properties of utomilumab and its variants and fragmentsare described in U.S. Pat. Nos. 8,821,867; 8,337,850; and 9,468,678, andInternational Patent Application Publication No. WO 2012/032433 A1, thedisclosures of each of which are incorporated by reference herein.Preclinical characteristics of utomilumab are described in Fisher, etal., Cancer Immunolog. & Immunother. 2012, 61, 1721-33. Current clinicaltrials of utomilumab in a variety of hematological and solid tumorindications include U.S. National Institutes of Healthclinicaltrials.gov identifiers NCT02444793, NCT01307267, NCT02315066,and NCT02554812.

In an embodiment, a 4-1BB agonist comprises a heavy chain given by SEQID NO:11 and a light chain given by SEQ ID NO:12. In an embodiment, a4-1BB agonist comprises heavy and light chains having the sequencesshown in SEQ ID NO:11 and SEQ ID NO:12, respectively, or antigen bindingfragments, Fab fragments, single-chain variable fragments (scFv),variants, or conjugates thereof. In an embodiment, a 4-1BB agonistcomprises heavy and light chains that are each at least 99% identical tothe sequences shown in SEQ ID NO:11 and SEQ ID NO:12, respectively. Inan embodiment, a 4-1BB agonist comprises heavy and light chains that areeach at least 98% identical to the sequences shown in SEQ ID NO:11 andSEQ ID NO:12, respectively. In an embodiment, a 4-1BB agonist comprisesheavy and light chains that are each at least 97% identical to thesequences shown in SEQ ID NO:11 and SEQ ID NO:12, respectively. In anembodiment, a 4-1BB agonist comprises heavy and light chains that areeach at least 96% identical to the sequences shown in SEQ ID NO:11 andSEQ ID NO:12, respectively. In an embodiment, a 4-1BB agonist comprisesheavy and light chains that are each at least 95% identical to thesequences shown in SEQ ID NO:11 and SEQ ID NO:12, respectively.

In an embodiment, the 4-1BB agonist comprises the heavy and light chainCDRs or variable regions (VRs) of utomilumab. In an embodiment, the4-1BB agonist heavy chain variable region (V_(H)) comprises the sequenceshown in SEQ ID NO: 13, and the 4-1BB agonist light chain variableregion (V_(L)) comprises the sequence shown in SEQ ID NO:14, andconservative amino acid substitutions thereof. In an embodiment, a 4-1BBagonist comprises V_(H) and V_(L) regions that are each at least 99%identical to the sequences shown in SEQ ID NO:13 and SEQ ID NO:14,respectively. In an embodiment, a 4-1BB agonist comprises V_(H) andV_(L) regions that are each at least 98% identical to the sequencesshown in SEQ ID NO:13 and SEQ ID NO:14, respectively. In an embodiment,a 4-1BB agonist comprises V_(H) and V_(L) regions that are each at least97% identical to the sequences shown in SEQ ID NO: 13 and SEQ ID NO:14,respectively. In an embodiment, a 4-1BB agonist comprises V_(H) andV_(L) regions that are each at least 96% identical to the sequencesshown in SEQ ID NO: 13 and SEQ ID NO:14, respectively. In an embodiment,a 4-1BB agonist comprises V_(H) and V_(L) regions that are each at least95% identical to the sequences shown in SEQ ID NO: 13 and SEQ ID NO:14,respectively. In an embodiment, a 4-1BB agonist comprises an scFvantibody comprising V_(H) and V_(L) regions that are each at least 99%identical to the sequences shown in SEQ ID NO:13 and SEQ ID NO:14.

In an embodiment, a 4-1BB agonist comprises heavy chain CDR1, CDR2 andCDR3 domains having the sequences set forth in SEQ ID NO: 15, SEQ IDNO:16, and SEQ ID NO:17, respectively, and conservative amino acidsubstitutions thereof, and light chain CDR1, CDR2 and CDR3 domainshaving the sequences set forth in SEQ ID NO:18, SEQ ID NO:19, and SEQ IDNO:20, respectively, and conservative amino acid substitutions thereof.

In an embodiment, the 4-1BB agonist is a 4-1BB agonist biosimilarmonoclonal antibody approved by drug regulatory authorities withreference to utomilumab. In an embodiment, the biosimilar monoclonalantibody comprises an 4-1BB antibody comprising an amino acid sequencewhich has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100%sequence identity, to the amino acid sequence of a reference medicinalproduct or reference biological product and which comprises one or morepost-translational modifications as compared to the reference medicinalproduct or reference biological product, wherein the reference medicinalproduct or reference biological product is utomilumab. In someembodiments, the one or more post-translational modifications areselected from one or more of glycosylation, oxidation, deamidation, andtruncation. In some embodiments, the biosimilar is a 4-1BB agonistantibody authorized or submitted for authorization, wherein the 4-BBagonist antibody is provided in a formulation which differs from theformulations of a reference medicinal product or reference biologicalproduct, wherein the reference medicinal product or reference biologicalproduct is utomilumab. The 4-1BB agonist antibody may be authorized by adrug regulatory authority such as the U.S. FDA and/or the EuropeanUnion's EMA. In some embodiments, the biosimilar is provided as acomposition which further comprises one or more excipients, wherein theone or more excipients are the same or different to the excipientscomprised in a reference medicinal product or reference biologicalproduct, wherein the reference medicinal product or reference biologicalproduct is utomilumab. In some embodiments, the biosimilar is providedas a composition which further comprises one or more excipients, whereinthe one or more excipients are the same or different to the excipientscomprised in a reference medicinal product or reference biologicalproduct, wherein the reference medicinal product or reference biologicalproduct is utomilumab.

TABLE 4Amino acid sequences for 4-1BB agonist antibodies related to utomilumab.Identifier Sequence (One-Letter Amino Acid Symbols) SEQ ID NO: 11EVQLVQSGAE VKKPGESLRI SCKGSGYSFS TYWISWVRQM PGKGLEWMGK IYPGDSYTNY  60heavy chain forSPSFQGQVTI SADKSISTAY LQWSSLKASD TAMYYCARGY GIFDYWGQGT LVTVSSASTK 120utomilumabGPSVFPLAPC SRSTSESTAA LGCLVKDYFP EPVTVSWNSG ALTSGVETFP AVLQSSGLYS 180LSSVVTVPSS NFGTQTYTCN VDHKPSNTKV DKTVERKCCV ECPPCPAPPV AGPSVFLFPP 240KPKDTLMISR TPEVTCVVVD VSHEDPEVQF NWYVDGVEVH NAKTKPREEQ FNSTFRVVSV 300LTVVHQDWLN GKEYKCKVSN KGLPAPIEKT ISKTKGQPRE PQVYTLPPSR EEMTKNQVSL 360TCLVKGFYPS DIAVEWESNG QPENNYKTTP PMLDSDGSFF LYSKLTVDKS RWQQGNVFSC 420SVMHEALHNH YTQKSLSLSP G                                           441SEQ ID NO: 12SYELTQPPSV SVSPGQTASI TCSGDNIGDQ YAHWYQQKPG QSPVLVIYQD KNRPSGIPER  60light chain forFSGSNSGNTA TLTISGTQAM DEADYYCATY TGFGSLAVFG GGTKLTVLGQ PKAAPSVTLF 120utomilumabPPSSEELQAN KATLVCLISD FYPGAVTVAW KADSSPVKAG VETTTPSKQS NNKYAASSYL 180SLTPEQWKSH RSYSCQVTHE GSTVEKTVAP TECS                             214SEQ ID NO: 13EVQLVQSGAE VKKPGESLRI SCKGSGYSFS TYWISWVRQM PGKGLEWMG KIYPGDSYTN   60heavy chainYSPSFQGQVT ISADKSISTA YLQWSSLKAS DTAMYYCARG YGIFDYWGQ GTLVTVSS    118variable region for utomilumab SEQ ID NO: 14SYELTQPPSV SVSPGQTASI TCSGDNIGDQ YAHWYQQKPG QSPVLVIYQD KNRPSGIPER  60light chainFSGSNSGNTA TLTISGTQAM DEADYYCATY TGFGSLAVFG GGTKLTVL              108variable region for utomilumab SEQ ID NO: 15STYWIS                                                              6heavy chain CDR1 for utomilumab SEQ ID NO: 16KIYPGDSYTN YSPSFQG                                                 17heavy chain CDR2 for utomilumab SEQ ID NO: 17RGYGIFDY                                                            8heavy chain CDR3 for utomilumab SEQ ID NO: 18SGDNIGDQYA H                                                       11light chain CDR1 for utomilumab SEQ ID NO: 19QDKNRPS                                                             7light chain CDR2 for utomilumab SEQ ID NO: 20ATYTGFGSLA V                                                       11light chain CDR3 for utomilumab

In a preferred embodiment, the 4-1BB agonist is the monoclonal antibodyurelumab, also known as BMS-663513 and 20H4.9.h4a, or a fragment,derivative, variant, or biosimilar thereof. Urelumab is available fromBristol-Myers Squibb, Inc., and Creative Biolabs, Inc. Urelumab is animmunoglobulin G4-kappa, anti-[Homo sapiens TNFRSF9 (tumor necrosisfactor receptor superfamily member 9, 4-1BB, T cell antigen ILA,CD137)], Homo sapiens (fully human) monoclonal antibody. The amino acidsequences of urelumab are set forth in Table 5. Urelumab comprisesN-glycosylation sites at positions 298 (and 298″); heavy chainintrachain disulfide bridges at positions 22-95 (V_(H)-V_(L)), 148-204(C_(H)1-C_(L)), 262-322 (C_(H)2) and 368-426 (C_(H)3) (and at positions22″-95″, 148″-204″, 262″-322″, and 368″-426″); light chain intrachaindisulfide bridges at positions 23′-88′ (V_(H)-V_(L)) and 136′-196′(C_(H)l-C_(L)) (and at positions 23′″-88′″ and 136′″-196′″); interchainheavy chain-heavy chain disulfide bridges at positions 227-227″ and230-230″; and interchain heavy chain-light chain disulfide bridges at135-216′ and 135″-216′″. The preparation and properties of urelumab andits variants and fragments are described in U.S. Pat. Nos. 7,288,638 and8,962,804, the disclosures of which are incorporated by referenceherein. The preclinical and clinical characteristics of urelumab aredescribed in Segal, et al., Clin. Cancer Res. 2016, available athttp:/dx.doi.org/10.1158/1078-0432.CCR-16-1272. Current clinical trialsof urelumab in a variety of hematological and solid tumor indicationsinclude U.S. National Institutes of Health clinicaltrials.govidentifiers NCT01775631, NCT02110082, NCT02253992, and NCT01471210.

In an embodiment, a 4-1BB agonist comprises a heavy chain given by SEQID NO:21 and a light chain given by SEQ ID NO:22. In an embodiment, a4-1BB agonist comprises heavy and light chains having the sequencesshown in SEQ ID NO:21 and SEQ ID NO:22, respectively, or antigen bindingfragments, Fab fragments, single-chain variable fragments (scFv),variants, or conjugates thereof. In an embodiment, a 4-1BB agonistcomprises heavy and light chains that are each at least 99% identical tothe sequences shown in SEQ ID NO:21 and SEQ ID NO:22, respectively. Inan embodiment, a 4-1BB agonist comprises heavy and light chains that areeach at least 98% identical to the sequences shown in SEQ ID NO:21 andSEQ ID NO:22, respectively. In an embodiment, a 4-1BB agonist comprisesheavy and light chains that are each at least 97% identical to thesequences shown in SEQ ID NO:21 and SEQ ID NO:22, respectively. In anembodiment, a 4-1BB agonist comprises heavy and light chains that areeach at least 96% identical to the sequences shown in SEQ ID NO:21 andSEQ ID NO:22, respectively. In an embodiment, a 4-1BB agonist comprisesheavy and light chains that are each at least 95% identical to thesequences shown in SEQ ID NO:21 and SEQ ID NO:22, respectively.

In an embodiment, the 4-1BB agonist comprises the heavy and light chainCDRs or variable regions (VRs) of urelumab. In an embodiment, the 4-1BBagonist heavy chain variable region (V_(H)) comprises the sequence shownin SEQ ID NO:23, and the 4-1BB agonist light chain variable region(V_(L)) comprises the sequence shown in SEQ ID NO:24, and conservativeamino acid substitutions thereof. In an embodiment, a 4-1BB agonistcomprises V_(H) and V_(L) regions that are each at least 99% identicalto the sequences shown in SEQ ID NO:23 and SEQ ID NO:24, respectively.In an embodiment, a 4-1BB agonist comprises V_(H) and V_(L) regions thatare each at least 98% identical to the sequences shown in SEQ ID NO:23and SEQ ID NO:24, respectively. In an embodiment, a 4-1BB agonistcomprises V_(H) and V_(L) regions that are each at least 97% identicalto the sequences shown in SEQ ID NO:23 and SEQ ID NO:24, respectively.In an embodiment, a 4-1BB agonist comprises V_(H) and V_(L) regions thatare each at least 96% identical to the sequences shown in SEQ ID NO:23and SEQ ID NO:24, respectively. In an embodiment, a 4-1BB agonistcomprises V_(H) and V_(L) regions that are each at least 95% identicalto the sequences shown in SEQ ID NO:23 and SEQ ID NO:24, respectively.In an embodiment, a 4-1BB agonist comprises an scFv antibody comprisingV_(H) and V_(L) regions that are each at least 99% identical to thesequences shown in SEQ ID NO:23 and SEQ ID NO:24.

In an embodiment, a 4-1BB agonist comprises heavy chain CDR1, CDR2 andCDR3 domains having the sequences set forth in SEQ ID NO:25, SEQ IDNO:26, and SEQ ID NO:27, respectively, and conservative amino acidsubstitutions thereof, and light chain CDR1, CDR2 and CDR3 domainshaving the sequences set forth in SEQ ID NO:28, SEQ ID NO:29, and SEQ IDNO:30, respectively, and conservative amino acid substitutions thereof.

In an embodiment, the 4-1BB agonist is a 4-1BB agonist biosimilarmonoclonal antibody approved by drug regulatory authorities withreference to urelumab. In an embodiment, the biosimilar monoclonalantibody comprises an 4-1BB antibody comprising an amino acid sequencewhich has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100%sequence identity, to the amino acid sequence of a reference medicinalproduct or reference biological product and which comprises one or morepost-translational modifications as compared to the reference medicinalproduct or reference biological product, wherein the reference medicinalproduct or reference biological product is urelumab. In someembodiments, the one or more post-translational modifications areselected from one or more of glycosylation, oxidation, deamidation, andtruncation. In some embodiments, the biosimilar is a 4-1BB agonistantibody authorized or submitted for authorization, wherein the 4-1BBagonist antibody is provided in a formulation which differs from theformulations of a reference medicinal product or reference biologicalproduct, wherein the reference medicinal product or reference biologicalproduct is urelumab. The 4-1BB agonist antibody may be authorized by adrug regulatory authority such as the U.S. FDA and/or the EuropeanUnion's EMA. In some embodiments, the biosimilar is provided as acomposition which further comprises one or more excipients, wherein theone or more excipients are the same or different to the excipientscomprised in a reference medicinal product or reference biologicalproduct, wherein the reference medicinal product or reference biologicalproduct is urelumab. In some embodiments, the biosimilar is provided asa composition which further comprises one or more excipients, whereinthe one or more excipients are the same or different to the excipientscomprised in a reference medicinal product or reference biologicalproduct, wherein the reference medicinal product or reference biologicalproduct is urelumab.

TABLE 5Amino acid sequences for 4-1BB agonist antibodies related to urelumab.Identifier Sequence (One-Letter Amino Acid Symbols) SEQ ID NO: 21QVQLQQWGAG LLKPSETLSL TCAVYGGSFS GYYWSWIRQS PEKGLEWIGE INHGGYVTYN  60heavy chain forPSLESRVTIS VDTSKNQFSL KLSSVTAADT AVYYCARDYG PGNYDWYFDL WGRGTLVTVS 120urelumabSASTKGPSVF PLAPCSRSTS ESTAALGCLV KDYFPEPVTV SWNSGALTSG VETFPAVIQS 180SGLYSLSSVV TVPSSSLGTK TYTCNVDHKP SNTKVEKRVE SKYGPPCPPC PAPEFLGGPS 240VFLFPPKPKD TLMISRTPEV TCVVVDVSQE DPEVQFNWYV DGVEVHNAKT KPREEQFNST 300YRVVSVLTVL HQDWLNGKEY KCKVSNKGLP SSIEKTISKA KGQPREPQVY TLPPSQEEMT 360KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSR LTVDKSRWQE 420GNVESCSVMH EALHNHYTQK SLSLSLGK                                    448SEQ ID NO: 22EIVLTQSPAT LSLSPGERAT LSCPASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA  60light chain forRFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPPALTF CGGTKVEIKR TVAAPSVFIF 120urelumabPPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN SQESVTEQDS KDSTYSLSST 180LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC                           216SEQ ID NO: 23MKHLWFFLLL VAAPRWVLSQ VQLQQWGAGL LKPSETLSLT CAVYGGSFSG YYWSWIRQSP  60variable heavyEKGLEWIGEI NHGGYVTYPP SLESRVTISV DTSKNQFSLK LSSVTAADTA VYYCARDYGP 120chain for urelumab SEQ ID NO: 24MEAPAQLLFL LLLWLPDTTG EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP  60variable lightGQAPRLLIYD ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ            110chain for urelumab SEQ ID NO: 25GYYWS                                                               5heavy chain CDR1 for urelumab SEQ ID NO: 26EINHGGYVTY NPSLES                                                  16heavy chain CDR2 for urelumab SEQ ID NO: 27DYGPGNYDWY FDL                                                     13heavy chain CDR3 for urelumab SEQ ID NO: 28RASQSVSSYL A                                                       11light chain CDR1 for urelumab SEQ ID NO: 29DASNRAT                                                             7light chain CDR2 for urelumab SEQ ID NO: 30QQRSDWPPAL T                                                       11light chain CDR3 for urelumab

In an embodiment, the 4-1BB agonist is selected from the groupconsisting of 1D8, 3Elor, 4B4 (BioLegend 309809), H4-1BB-M127 (BDPharmingen 552532), BBK2 (Thermo Fisher MS621PABX), 145501 (LeincoTechnologies B591), the antibody produced by cell line deposited as ATCCNo. HB-11248 and disclosed in U.S. Pat. No. 6,974,863, 5F4 (BioLegend 311503), C65-485 (BD Pharmingen 559446), antibodies disclosed in U.S.Patent Application Publication No. US 2005/0095244, antibodies disclosedin U.S. Pat. No. 7,288,638 (such as 20H4.9-IgG1 (BMS-663031)),antibodies disclosed in U.S. Pat. No. 6,887,673 (such as 4E9 orBMS-554271), antibodies disclosed in U.S. Pat. No. 7,214,493, antibodiesdisclosed in U.S. Pat. No. 6,303,121, antibodies disclosed in U.S. Pat.No. 6,569,997, antibodies disclosed in U.S. Pat. No. 6,905,685 (such as4E9 or BMS-554271), antibodies disclosed in U.S. Pat. No. 6,362,325(such as 1D8 or BMS-469492; 3H3 or BMS-469497; or 3E1), antibodiesdisclosed in U.S. Pat. No. 6,974,863 (such as 53A2); antibodiesdisclosed in U.S. Pat. No. 6,210,669 (such as 1D8, 3B8, or 3E1),antibodies described in U.S. Pat. No. 5,928,893, antibodies disclosed inU.S. Pat. No. 6,303,121, antibodies disclosed in U.S. Pat. No.6,569,997, antibodies disclosed in International Patent ApplicationPublication Nos. WO 2012/177788, WO 2015/119923, and WO 2010/042433, andfragments, derivatives, conjugates, variants, or biosimilars thereof,wherein the disclosure of each of the foregoing patents or patentapplication publications is incorporated by reference here.

In an embodiment, the 4-1BB agonist is a 4-1BB agonistic fusion proteindescribed in International Patent Application Publication Nos. WO2008/025516 A1, WO 2009/007120 A1, WO 2010/003766 A1, WO 2010/010051 A1,and WO 2010/078966 A1; U.S. Patent Application Publication Nos. US2011/0027218 A1, US 2015/0126709 A1, US 2011/0111494 A1, US 2015/0110734A1, and US 2015/0126710 A1; and U.S. Pat. Nos. 9,359,420, 9,340,599,8,921,519, and 8,450,460, the disclosures of which are incorporated byreference herein.

In an embodiment, the 4-1BB agonist is a 4-1BB agonistic fusion proteinas depicted in Structure I-A (C-terminal Fc-antibody fragment fusionprotein) or Structure I-B (N-terminal Fc-antibody fragment fusionprotein) of FIG. 22, or a fragment, derivative, conjugate, variant, orbiosimilar thereof.

In structures I-A and I-B of FIG. 22, the cylinders refer to individualpolypeptide binding domains. Structures I-A and I-B comprise threelinearly-linked TNFRSF binding domains derived from e.g., 4-1BBL or anantibody that binds 4-1BB, which fold to form a trivalent protein, whichis then linked to a second trivalent protein through IgG1-Fc (includingC_(H)3 and C_(H)2 domains) is then used to link two of the trivalentproteins together through disulfide bonds (small elongated ovals),stabilizing the structure and providing an agonists capable of bringingtogether the intracellular signaling domains of the six receptors andsignaling proteins to form a signaling complex. The TNFRSF bindingdomains denoted as cylinders may be scFv domains comprising, e.g., aV_(H) and a V_(L) chain connected by a linker that may comprisehydrophilic residues and Gly and Ser sequences for flexibility, as wellas Glu and Lys for solubility. Any scFv domain design may be used, suchas those described in de Marco, Microbial Cell Factories, 2011, 10, 44;Ahmad, et al., Clin. & Dev. Immunol. 2012, 980250; Monnier, et al.,Antibodies, 2013, 2, 193-208; or in references incorporated elsewhereherein. Fusion protein structures of this form are described in U.S.Pat. Nos. 9,359,420, 9,340,599, 8,921,519, and 8,450,460, thedisclosures of which are incorporated by reference herein.

Amino acid sequences for the other polypeptide domains of structure I-Aare given in Table 6. The Fc domain preferably comprises a completeconstant domain (amino acids 17-230 of SEQ ID NO:31) the complete hingedomain (amino acids 1-16 of SEQ ID NO:31) or a portion of the hingedomain (e.g., amino acids 4-16 of SEQ ID NO:31). Preferred linkers forconnecting a C-terminal Fc-antibody may be selected from the embodimentsgiven in SEQ ID NO:32 to SEQ ID NO:41, including linkers suitable forfusion of additional polypeptides.

TABLE 6Amino acid sequences for TNFRSF fusion proteins, including 4-1BB fusion proteins,with C-terminal Fc-antibody fragment fusion protein design (structure I-A).Identifier Sequence (One-Letter Amino Acid Symbols) SEQ ID NO: 31KSCDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW  60Fc domainYVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS 120KAKGQPREPQ VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV 180LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK            230SEQ ID NO: 32GGPGSSKSCD KTHTCPPCPA PE                                           22linker SEQ ID NO: 33GGSGSSKSCD KTHTCPPCPA PE                                           22linker SEQ ID NO: 34GGPGSSSSSS SKSCDKTHTC PPCPAPE                                      27linker SEQ ID NO: 35GGSGSSSSSS SKSCDKTHTC PPCPAPE                                      27linker SEQ ID NO: 36GGPGSSSSSS SSSKSCDKTH TCPPCPAPE                                    29linker SEQ ID NO: 37GGSGSSSSSS SSSKSCDKTH TCPPCPAPE                                    29linker SEQ ID NO: 38GGPGSSGSGS SDKTHTCPPC PAPE                                         24linker SEQ ID NO: 39GGPGSSGSGS DKTHTCPPCP APE                                          23linker SEQ ID NO: 40GGPSSSGSDK THTCPPCPAP E                                            21linker SEQ ID NO: 41GGSSSSSSSS GSDKTHTCPP CPAPE                                        25linker

Amino acid sequences for the other polypeptide domains of structure I-Bare given in Table 7. If an Fc antibody fragment is fused to theN-terminus of an TNRFSF fusion protein as in structure I-B, the sequenceof the Fc module is preferably that shown in SEQ ID NO:42, and thelinker sequences are preferably selected from those embodiments setforth in SEQ ID NO:43 to SEQ ID NO:45.

TABLE 7 Amino acid sequences for TNFRSF fusion proteins,including 4-1BB fusion proteins, with N-terminalFc-antibody fragment fusion protein design (structure I-B). IdentifierSequence (One-Letter Amino Acid Symbols) SEQ ID NO: 42METDTLLLWV LLLWVPAGNG DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT  60Fc domainCVVVDVSHED PEVKFNWYVD GVEVENAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK 120CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE 180WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS 240LSLSPG                                                            246SEQ ID NO: 43SGSGSGSGSG S                                                       11linker SEQ ID NO: 44SSSSSSGSGS GS                                                      12linker SEQ ID NO: 45SSSSSSGSGS GSGSGS                                                  16linker

In an embodiment, a 4-1BB agonist fusion protein according to structuresI-A or I-B of FIG. 22 comprises one or more 4-1BB binding domainsselected from the group consisting of a variable heavy chain andvariable light chain of utomilumab, a variable heavy chain and variablelight chain of urelumab, a variable heavy chain and variable light chainof utomilumab, a variable heavy chain and variable light chain selectedfrom the variable heavy chains and variable light chains described inTable 8, any combination of a variable heavy chain and variable lightchain of the foregoing, and fragments, derivatives, conjugates,variants, and biosimilars thereof.

In an embodiment, a 4-1BB agonist fusion protein according to structuresI-A or I-B of FIG. 22 comprises one or more 4-1BB binding domainscomprising a 4-1BBL sequence. In an embodiment, a 4-1BB agonist fusionprotein according to structures I-A or I-B comprises one or more 4-1BBbinding domains comprising a sequence according to SEQ ID NO:46. In anembodiment, a 4-1BB agonist fusion protein according to structures I-Aor I-B comprises one or more 4-1BB binding domains comprising a soluble4-1BBL sequence. In an embodiment, a 4-1BB agonist fusion proteinaccording to structures I-A or I-B comprises one or more 4-1BB bindingdomains comprising a sequence according to SEQ ID NO:47.

In an embodiment, a 4-1BB agonist fusion protein according to structuresI-A or I-B of FIG. 22 comprises one or more 4-1BB binding domains thatis a scFv domain comprising V_(H) and V_(L) regions that are each atleast 95% identical to the sequences shown in SEQ ID NO:13 and SEQ IDNO: 14, respectively, wherein the V_(H) and V_(L) domains are connectedby a linker. In an embodiment, a 4-1BB agonist fusion protein accordingto structures I-A or I-B comprises one or more 4-1BB binding domainsthat is a scFv domain comprising V_(H) and V_(L) regions that are eachat least 95% identical to the sequences shown in SEQ ID NO:23 and SEQ IDNO:24, respectively, wherein the V_(H) and V_(L) domains are connectedby a linker. In an embodiment, a 4-1BB agonist fusion protein accordingto structures I-A or I-B comprises one or more 4-1BB binding domainsthat is a scFv domain comprising V_(H) and V_(L) regions that are eachat least 95% identical to the V_(H) and V_(L) sequences given in Table8, wherein the V_(H) and V_(L) domains are connected by a linker.

TABLE 8 Additional polypeptide domains useful as 4-1BB binding domainsin fusion proteins or as scFy 4-1BB agonist antibodies. IdentifierSequence (One-Letter Amino Acid Symbols) SEQ ID NO: 46MEYASDASLD PEAPWPPAPR ARACRVLPWA LVAGLLLLLL LAAACAVFLA CPWAVSGARA  604-1BBLSPGSAASPRL REGPELSPDD PAGLLDLRQG MFAQLVAQNV LLIDGPLSWY SDPGLAGVSL 120TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVVAGEGSGS VSLALHLQPL RSAAGAAALA 180LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWQLTQ GATVLGLFRV 240TPEIPAGLPS PRSE                                                   254SEQ ID NO: 47LRQGMFAQLV AQNVLLIDGP LSWYSDPGLA GVSLTGGLSY KEDTKELVVA KAGVYYVFFQ  604-1BBL solubleLELRRVVAGE GSGSVSLALH LQPLRSAAGA AALALTVDLP PASSEARNSA FGFQGRLLHL 120domainSAGQRLGVEL HTEARARHAW QLTQGATVLG LFRVTPEIPA GLPSPRSE              168SEQ ID NO: 48QVQLQQPGAE LVKPGASVKL SCKASGYTFS SYWMHWVKQR PGQVLEWIGE INPGNGHTNY  60variable heavyNEKFKSKATL TVDKSSSTAY MQLSSLTSED SAVYYCARSF TTARGFAYWG QGTLVTVS   118chain for 4B4-1-1 version 1 SEQ ID NO: 49DIVMTQSPATQSVTPGDRVS LSCPASQTIS DYLHWYQQKS HESPRLLIKY ASQSISGIPS   60variable lightRFSGSGSGSD FTLSINSVEP EDVGVYYCQD GHSFPPTFGG GTKLEIK               107chain for 4B4-1-1 version 1 SEQ ID NO: 50QVQLQQPGAE LVKPGASVKL SCKASGYTFS SYWMHWVKQR PGQVLEWIGE INPGNGHTNY  60variable heavyNEKFKSKATL TVDKSSSTAY MQLSSLTSED SAVYYCARSF TTARGFAYWG QGTLVTVSA  119chain for 4B4-1-1 version 2 SEQ ID NO: 51DIVMTQSPAT QSVTPGDRVS LSCPASQTIS DYLHWYQQKS HESPRLLIKY ASQSISGIPS  60variable lightRFSGSGSGSD FTLSINSVEP EDVGVYYCQD GHSFPPTFGG GTKLEIKR              108chain for 4B4-1-1 version 2 SEQ ID NO: 52MDWTWRILFL VAAATGAHSE VQLVESGGGL VQPGGSLRLS CAASGFTFSD YWMSWVRQAP  60variable heavyGKGLEWVADI KNDGSYTNYA PSLTNRFTIS RDNAKNSLYL QMNSLPAEDT AVYYCARELT 120chain for H39E3-2 SEQ ID NO: 53MEAPAQLLFL LLLWLPDTTG DIVMTQSPDS LAVSLGERAT INCKSSQSLL SSGNQKNYL   60variable lightWYQQKPGQPP KLLIYYASTR QSGVPDRFSG SGSGTDFTLT ISSLQAEDVA            110chain for H39E3-2

In an embodiment, the 4-1BB agonist is a 4-1BB agonistic single-chainfusion polypeptide comprising (i) a first soluble 4-1BB binding domain,(ii) a first peptide linker, (iii) a second soluble 4-1BB bindingdomain, (iv) a second peptide linker, and (v) a third soluble 4-1BBbinding domain, further comprising an additional domain at theN-terminal and/or C-terminal end, and wherein the additional domain is aFab or Fc fragment domain. In an embodiment, the 4-1BB agonist is a4-1BB agonistic single-chain fusion polypeptide comprising (i) a firstsoluble 4-1BB binding domain, (ii) a first peptide linker, (iii) asecond soluble 4-1BB binding domain, (iv) a second peptide linker, and(v) a third soluble 4-1BB binding domain, further comprising anadditional domain at the N-terminal and/or C-terminal end, wherein theadditional domain is a Fab or Fc fragment domain, wherein each of thesoluble 4-1BB domains lacks a stalk region (which contributes totrimerisation and provides a certain distance to the cell membrane, butis not part of the 4-1BB binding domain) and the first and the secondpeptide linkers independently have a length of 3-8 amino acids.

In an embodiment, the 4-1BB agonist is a 4-1BB agonistic single-chainfusion polypeptide comprising (i) a first soluble tumor necrosis factor(TNF) superfamily cytokine domain, (ii) a first peptide linker, (iii) asecond soluble TNF superfamily cytokine domain, (iv) a second peptidelinker, and (v) a third soluble TNF superfamily cytokine domain, whereineach of the soluble TNF superfamily cytokine domains lacks a stalkregion and the first and the second peptide linkers independently have alength of 3-8 amino acids, and wherein each TNF superfamily cytokinedomain is a 4-1BB binding domain.

In an embodiment, the 4-1BB agonist is a 4-1BB agonistic scFv antibodycomprising any of the foregoing V_(H) domains linked to any of theforegoing V_(L) domains.

In an embodiment, the 4-1BB agonist is BPS Bioscience 4-1BB agonistantibody catalog no. 79097-2, commercially available from BPSBioscience, San Diego, Calif., USA. In an embodiment, the 4-1BB agonistis Creative Biolabs 4-1BB agonist antibody catalog no. MOM-18179,commercially available from Creative Biolabs, Shirley, N.Y., USA.

The OX40 (CD134) agonist may be any OX40 binding molecule known in theart. The OX40 binding molecule may be a monoclonal antibody or fusionprotein capable of binding to human or mammalian OX40. The OX40 agonistsor OX40 binding molecules may comprise an immunoglobulin heavy chain ofany isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1,IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.The OX40 agonist or OX40 binding molecule may have both a heavy and alight chain. As used herein, the term binding molecule also includesantibodies (including full length antibodies), monoclonal antibodies(including full length monoclonal antibodies), polyclonal antibodies,multispecific antibodies (e.g., bispecific antibodies), human, humanizedor chimeric antibodies, and antibody fragments, e.g., Fab fragments,F(ab′) fragments, fragments produced by a Fab expression library,epitope-binding fragments of any of the above, and engineered forms ofantibodies, e.g., scFv molecules, that bind to OX40. In an embodiment,the OX40 agonist is an antigen binding protein that is a fully humanantibody. In an embodiment, the OX40 agonist is an antigen bindingprotein that is a humanized antibody. In some embodiments, OX40 agonistsfor use in the presently disclosed methods and compositions includeanti-OX40 antibodies, human anti-OX40 antibodies, mouse anti-OX40antibodies, mammalian anti-OX40 antibodies, monoclonal anti-OX40antibodies, polyclonal anti-OX40 antibodies, chimeric anti-OX40antibodies, anti-OX40 adnectins, anti-OX40 domain antibodies, singlechain anti-OX40 fragments, heavy chain anti-OX40 fragments, light chainanti-OX40 fragments, anti-OX40 fusion proteins, and fragments,derivatives, conjugates, variants, or biosimilars thereof. In apreferred embodiment, the OX40 agonist is an agonistic, anti-OX40humanized or fully human monoclonal antibody (i.e., an antibody derivedfrom a single cell line).

In a preferred embodiment, the OX40 agonist or OX40 binding molecule mayalso be a fusion protein. OX40 fusion proteins comprising an Fc domainfused to OX40L are described, for example, in Sadun, et al., JImmunother. 2009, 182, 1481-89. In a preferred embodiment, a multimericOX40 agonist, such as a trimeric or hexameric OX40 agonist (with threeor six ligand binding domains), may induce superior receptor (OX40L)clustering and internal cellular signaling complex formation compared toan agonistic monoclonal antibody, which typically possesses two ligandbinding domains. Trimeric (trivalent) or hexameric (or hexavalent) orgreater fusion proteins comprising three TNFRSF binding domains andIgG1-Fc and optionally further linking two or more of these fusionproteins are described, e.g., in Gieffers, et al., Mol. CancerTherapeutics 2013, 12, 2735-47.

Agonistic OX40 antibodies and fusion proteins are known to induce strongimmune responses. Curti, et al., Cancer Res. 2013, 73, 7189-98. In apreferred embodiment, the OX40 agonist is a monoclonal antibody orfusion protein that binds specifically to OX40 antigen in a mannersufficient to reduce toxicity. In some embodiments, the OX40 agonist isan agonistic OX40 monoclonal antibody or fusion protein that abrogatesantibody-dependent cellular toxicity (ADCC), for example NK cellcytotoxicity. In some embodiments, the OX40 agonist is an agonistic OX40monoclonal antibody or fusion protein that abrogates antibody-dependentcell phagocytosis (ADCP). In some embodiments, the OX40 agonist is anagonistic OX40 monoclonal antibody or fusion protein that abrogatescomplement-dependent cytotoxicity (CDC). In some embodiments, the OX40agonist is an agonistic OX40 monoclonal antibody or fusion protein whichabrogates Fc region functionality.

In some embodiments, the OX40 agonists are characterized by binding tohuman OX40 (SEQ ID NO:54) with high affinity and agonistic activity. Inan embodiment, the OX40 agonist is a binding molecule that binds tohuman OX40 (SEQ ID NO:54). In an embodiment, the OX40 agonist is abinding molecule that binds to murine OX40 (SEQ ID NO:55). The aminoacid sequences of OX40 antigen to which an OX40 agonist or bindingmolecule binds are summarized in Table 9.

TABLE 9 Amino acid sequences of OX40 antigens. IdentifierSequence (One-Letter Amino Acid Symbols) SEQ ID NO: 54MCVGARRLGR GPCAALLLLG LGLSTVTGLH CVGDTYPSND RCCHECRPGN GMVSRCSRSQ  60human OX40NTVCRPCGPG FYNDVVSSKP CKPCTWCNLR SGSERKQLCT ATQDTVCRCR AGTQPLDSYK 120(Homo sapiens)PGVDCAPCPP GHFSPGDNQA CKPWTNCTLA GKHTLQPASN SSDAICEDRD PPATQPQETQ 180GPPARPITVQ PTEAWPRTSQ GPSTRPVEVP GGRAVAAILG LGLVLGLLGP LAILLALYLL 240RRDQRLPPDA HKPPGGGSFR TPIQEEQADA HSTLAKI                          277SEQ ID NO: 55MYVWVQQPTA LLLLGLTLGV TARRLNCVKH TYPSGHKCCR ECQPGHGMVS RCDHTRDTLC  60murine OX40HPCETGFYNE AVNYDTCKQC TQCNHRSGSE LKQNCTPTQD TVCRCRPGTQ PRQDSGYKLG 120(Mus musculus)VDCVPCPPGH FSPGNNQACK PWTNCTLSGK QTRHPASDSL DAVCEDRSLL ATLLWETQRP 180TFRPTTVQST TVWPRTSELP SPPTLVTPEG PAFAVILGLG LGLLAPLTVL LALYLLRKAW 240RLPNTPKPCW GNSFRTPIQE EHTDAHFTLA KI                               272

In some embodiments, the compositions, processes and methods describedinclude a OX40 agonist that binds human or murine OX40 with a K_(D) ofabout 100 μM or lower, binds human or murine OX40 with a K_(D) of about90 μM or lower, binds human or murine OX40 with a K_(D) of about 80 μMor lower, binds human or murine OX40 with a K_(D) of about 70 μM orlower, binds human or murine OX40 with a K_(D) of about 60 μM or lower,binds human or murine OX40 with a K_(D) of about 50 μM or lower, bindshuman or murine OX40 with a K_(D) of about 40 μM or lower, or bindshuman or murine OX40 with a K_(D) of about 30 μM or lower.

In some embodiments, the compositions, processes and methods describedinclude a OX40 agonist that binds to human or murine OX40 with ak_(assoc) of about 7.5×10⁵ l/M·s or faster, binds to human or murineOX40 with a k_(assoc) of about 7.5×10⁵ l/M·s or faster, binds to humanor murine OX40 with a k_(assoc) of about 8×10⁵ l/M·s or faster, binds tohuman or murine OX40 with a k_(assoc) of about 8.5×10⁵ l/M·s or faster,binds to human or murine OX40 with a k_(assoc) of about 9×10⁵ l/M·s orfaster, binds to human or murine OX40 with a k_(assoc) of about 9.5×10⁵l/M·s or faster, or binds to human or murine OX40 with a k_(assoc) ofabout 1×10⁶ l/M·s or faster.

In some embodiments, the compositions, processes and methods describedinclude a OX40 agonist that binds to human or murine OX40 with ak_(dissoc) of about 2×10⁻⁵ l/s or slower, binds to human or murine OX40with a k_(dissoc) of about 2.1×10⁻⁵ l/s or slower, binds to human ormurine OX40 with a k_(dissoc) of about 2.2×10⁻⁵ l/s or slower, binds tohuman or murine OX40 with a k_(dissoc) of about 2.3×10⁻⁵ l/s or slower,binds to human or murine OX40 with a k_(dissoc) of about 2.4×10⁻⁵ l/s orslower, binds to human or murine OX40 with a k_(dissoc) of about2.5×10⁻⁵ l/s or slower, binds to human or murine OX40 with a k_(dissoc)of about 2.6×10⁻⁵ l/s or slower or binds to human or murine OX40 with ak_(dissoc) of about 2.7×10⁻⁵ l/s or slower, binds to human or murineOX40 with a k_(dissoc) of about 2.8×10⁻⁵ l/s or slower, binds to humanor murine OX40 with a k_(dissoc) of about 2.9×10⁻⁵ l/s or slower, orbinds to human or murine OX40 with a k_(dissoc) of about 3×10⁻⁵ l/s orslower.

In some embodiments, the compositions, processes and methods describedinclude OX40 agonist that binds to human or murine OX40 with an IC₅₀ ofabout 10 nM or lower, binds to human or murine OX40 with an IC₅₀ ofabout 9 nM or lower, binds to human or murine OX40 with an IC₅₀ of about8 nM or lower, binds to human or murine OX40 with an IC₅₀ of about 7 nMor lower, binds to human or murine OX40 with an IC₅₀ of about 6 nM orlower, binds to human or murine OX40 with an IC₅₀ of about 5 nM orlower, binds to human or murine OX40 with an IC₅₀ of about 4 nM orlower, binds to human or murine OX40 with an IC₅₀ of about 3 nM orlower, binds to human or murine OX40 with an IC₅₀ of about 2 nM orlower, or binds to human or murine OX40 with an IC₅₀ of about 1 nM orlower.

In some embodiments, the OX40 agonist is tavolixizumab, also known asMEDI0562 or MEDI-0562. Tavolixizumab is available from the MedImmunesubsidiary of AstraZeneca, Inc. Tavolixizumab is immunoglobulinG1-kappa, anti-[Homo sapiens TNFRSF4 (tumor necrosis factor receptor(TNFR) superfamily member 4, OX40, CD134)], humanized and chimericmonoclonal antibody. The amino acid sequences of tavolixizumab are setforth in Table 10. Tavolixizumab comprises N-glycosylation sites atpositions 301 and 301″, with fucosylated complex bi-antennary CHO-typeglycans; heavy chain intrachain disulfide bridges at positions 22-95(V_(H)-V_(L)), 148-204 (C_(H)1-C_(L)), 265-325 (C_(H)2) and 371-429(C_(H)3) (and at positions 22″-95″, 148″-204″, 265″-325″, and371″-429″); light chain intrachain disulfide bridges at positions23′-88′ (V_(H)-V_(L)) and 134′-194′ (C_(H)1-C_(L)) (and at positions23′″-88′″ and 134′″-194′″); interchain heavy chain-heavy chain disulfidebridges at positions 230-230″ and 233-233″; and interchain heavychain-light chain disulfide bridges at 224-214′ and 224″-214′″. Currentclinical trials of tavolixizumab in a variety of solid tumor indicationsinclude U.S. National Institutes of Health clinicaltrials.govidentifiers NCT02318394 and NCT02705482.

In an embodiment, a OX40 agonist comprises a heavy chain given by SEQ IDNO:56 and a light chain given by SEQ ID NO:57. In an embodiment, a OX40agonist comprises heavy and light chains having the sequences shown inSEQ ID NO:56 and SEQ ID NO:57, respectively, or antigen bindingfragments, Fab fragments, single-chain variable fragments (scFv),variants, or conjugates thereof. In an embodiment, a OX40 agonistcomprises heavy and light chains that are each at least 99% identical tothe sequences shown in SEQ ID NO:56 and SEQ ID NO:57, respectively. Inan embodiment, a OX40 agonist comprises heavy and light chains that areeach at least 98% identical to the sequences shown in SEQ ID NO:56 andSEQ ID NO:57, respectively. In an embodiment, a OX40 agonist comprisesheavy and light chains that are each at least 97% identical to thesequences shown in SEQ ID NO:56 and SEQ ID NO:57, respectively. In anembodiment, a OX40 agonist comprises heavy and light chains that areeach at least 96% identical to the sequences shown in SEQ ID NO:56 andSEQ ID NO:57, respectively. In an embodiment, a OX40 agonist comprisesheavy and light chains that are each at least 95% identical to thesequences shown in SEQ ID NO:56 and SEQ ID NO:57, respectively.

In an embodiment, the OX40 agonist comprises the heavy and light chainCDRs or variable regions (VRs) of tavolixizumab. In an embodiment, theOX40 agonist heavy chain variable region (V_(H)) comprises the sequenceshown in SEQ ID NO:58, and the OX40 agonist light chain variable region(V_(L)) comprises the sequence shown in SEQ ID NO:59, and conservativeamino acid substitutions thereof. In an embodiment, a OX40 agonistcomprises V_(H) and V_(L) regions that are each at least 99% identicalto the sequences shown in SEQ ID NO:58 and SEQ ID NO:59, respectively.In an embodiment, a OX40 agonist comprises V_(H) and V_(L) regions thatare each at least 98% identical to the sequences shown in SEQ ID NO:58and SEQ ID NO:59, respectively. In an embodiment, a OX40 agonistcomprises V_(H) and V_(L) regions that are each at least 97% identicalto the sequences shown in SEQ ID NO:58 and SEQ ID NO:59, respectively.In an embodiment, a OX40 agonist comprises V_(H) and V_(L) regions thatare each at least 96% identical to the sequences shown in SEQ ID NO:58and SEQ ID NO:59, respectively. In an embodiment, a OX40 agonistcomprises V_(H) and V_(L) regions that are each at least 95% identicalto the sequences shown in SEQ ID NO:58 and SEQ ID NO:59, respectively.In an embodiment, an OX40 agonist comprises an scFv antibody comprisingV_(H) and V_(L) regions that are each at least 99% identical to thesequences shown in SEQ ID NO:58 and SEQ ID NO:59.

In an embodiment, a OX40 agonist comprises heavy chain CDR1, CDR2 andCDR3 domains having the sequences set forth in SEQ ID NO:60, SEQ IDNO:61, and SEQ ID NO:62, respectively, and conservative amino acidsubstitutions thereof, and light chain CDR1, CDR2 and CDR3 domainshaving the sequences set forth in SEQ ID NO:63, SEQ ID NO:64, and SEQ IDNO:65, respectively, and conservative amino acid substitutions thereof.

In an embodiment, the OX40 agonist is a OX40 agonist biosimilarmonoclonal antibody approved by drug regulatory authorities withreference to tavolixizumab. In an embodiment, the biosimilar monoclonalantibody comprises an OX40 antibody comprising an amino acid sequencewhich has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100%sequence identity, to the amino acid sequence of a reference medicinalproduct or reference biological product and which comprises one or morepost-translational modifications as compared to the reference medicinalproduct or reference biological product, wherein the reference medicinalproduct or reference biological product is tavolixizumab. In someembodiments, the one or more post-translational modifications areselected from one or more of: glycosylation, oxidation, deamidation, andtruncation. In some embodiments, the biosimilar is a OX40 agonistantibody authorized or submitted for authorization, wherein the OX40agonist antibody is provided in a formulation which differs from theformulations of a reference medicinal product or reference biologicalproduct, wherein the reference medicinal product or reference biologicalproduct is tavolixizumab. The OX40 agonist antibody may be authorized bya drug regulatory authority such as the U.S. FDA and/or the EuropeanUnion's EMA. In some embodiments, the biosimilar is provided as acomposition which further comprises one or more excipients, wherein theone or more excipients are the same or different to the excipientscomprised in a reference medicinal product or reference biologicalproduct, wherein the reference medicinal product or reference biologicalproduct is tavolixizumab. In some embodiments, the biosimilar isprovided as a composition which further comprises one or moreexcipients, wherein the one or more excipients are the same or differentto the excipients comprised in a reference medicinal product orreference biological product, wherein the reference medicinal product orreference biological product is tavolixizumab.

TABLE 10Amino acid sequences for OX40 agonist antibodies related to tavolixizumab.Identifier Sequence (One-Letter Amino Acid Symbols) SEQ ID NO: 56QVQLQESGPG LVKPSQTLSL TCAVYGGSFS SGYWNWIRKH PGKGLEYIGY ISYNGITYHN  60heavy chain forPSLKSRITIN RDTSKNQYSL QLNSVTPEDT AVYYCARYKY DYDGGHAMDY WGQGTLVTVS 120tavolixizumabSASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV SWNSGALTSG VETFPAVIQS 180SGLYSLSSVV TVPSSSLGTQ TYICNVNHKP SNTKVDKRVE PKSCDKTHTC PPCPAPELLG 240GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY 300NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPSRE 360EMTKNQVSLT CLVNGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSKLTVDKSR 420WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K                                451SEQ ID NO: 57DIQMTQSPSS LSASVGDRVT ITCPASQDIS NYLNWYQQKP GKAPKLLIYY TSKLHSGVPS  60light chain forRFSGSGSGTD YTLTISSLQP EDFATYYCQQ GSALPWTFGQ GTKVEIKRTV AAPSVFIFPP 120tavolixizumabSDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT 180LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC                             214SEQ ID NO: 58QVQLQESGPG LVKPSQTLSL TCAVYGGSFS SGYWNWIRKH PGKGLEYIGY ISYNGITYHN  60heavy chainPSLKSRITIN RDTSKNQYSL QLNSVTPEDT AVYYCARYKY DYDGGHAMDY WGQGTLVT   118variable region for tavolixizumab SEQ ID NO: 59DIQMTQSPSS LSASVGDRVT ITCPASQDIS NYLNWYQQKP GKAPKLLIYY TSKLHSGVPS  60light chainRFSGSGSGTD YTLTISSLQP EDFATYYCQQ GSALPWTFGQ GTKVEIKR              108variable region for tavolixizumab SEQ ID NO: 60GSFSSGYWN                                                           9heavy chain CDR1 for tavolixizumab SEQ ID NO: 61YIGYISYNGI TYH                                                     13heavy chain CDR2 for tavolixizumab SEQ ID NO: 62RYKYDYDGGH AMDY                                                    14heavy chain CDR3 for tavolixizumab SEQ ID NO: 63QDISNYLN                                                            8light chain CDR1 for tavolixizumab SEQ ID NO: 64LLIYYTSKLH S                                                       11light chain CDR2 for tavolixizumab SEQ ID NO: 65QQGSALPW                                                            8light chain CDR3 for tavolixizumab

In some embodiments, the OX40 agonist is 11D4, which is a fully humanantibody available from Pfizer, Inc. The preparation and properties of11D4 are described in U.S. Pat. Nos. 7,960,515; 8,236,930; and9,028,824, the disclosures of which are incorporated by referenceherein. The amino acid sequences of 11D4 are set forth in Table 11.

In an embodiment, a OX40 agonist comprises a heavy chain given by SEQ IDNO:66 and a light chain given by SEQ ID NO:67. In an embodiment, a OX40agonist comprises heavy and light chains having the sequences shown inSEQ ID NO:66 and SEQ ID NO:67, respectively, or antigen bindingfragments, Fab fragments, single-chain variable fragments (scFv),variants, or conjugates thereof. In an embodiment, a OX40 agonistcomprises heavy and light chains that are each at least 99% identical tothe sequences shown in SEQ ID NO:66 and SEQ ID NO:67, respectively. Inan embodiment, a OX40 agonist comprises heavy and light chains that areeach at least 98% identical to the sequences shown in SEQ ID NO:66 andSEQ ID NO:67, respectively. In an embodiment, a OX40 agonist comprisesheavy and light chains that are each at least 97% identical to thesequences shown in SEQ ID NO:66 and SEQ ID NO:67, respectively. In anembodiment, a OX40 agonist comprises heavy and light chains that areeach at least 96% identical to the sequences shown in SEQ ID NO:66 andSEQ ID NO:67, respectively. In an embodiment, a OX40 agonist comprisesheavy and light chains that are each at least 95% identical to thesequences shown in SEQ ID NO:66 and SEQ ID NO:67, respectively.

In an embodiment, the OX40 agonist comprises the heavy and light chainCDRs or variable regions (VRs) of 11D4. In an embodiment, the OX40agonist heavy chain variable region (V_(H)) comprises the sequence shownin SEQ ID NO:68, and the OX40 agonist light chain variable region(V_(L)) comprises the sequence shown in SEQ ID NO:69, and conservativeamino acid substitutions thereof. In an embodiment, a OX40 agonistcomprises V_(H) and V_(L) regions that are each at least 99% identicalto the sequences shown in SEQ ID NO:68 and SEQ ID NO:69, respectively.In an embodiment, a OX40 agonist comprises V_(H) and V_(L) regions thatare each at least 98% identical to the sequences shown in SEQ ID NO:68and SEQ ID NO:69, respectively. In an embodiment, a OX40 agonistcomprises V_(H) and V_(L) regions that are each at least 97% identicalto the sequences shown in SEQ ID NO:68 and SEQ ID NO:69, respectively.In an embodiment, a OX40 agonist comprises V_(H) and V_(L) regions thatare each at least 96% identical to the sequences shown in SEQ ID NO:68and SEQ ID NO:69, respectively. In an embodiment, a OX40 agonistcomprises V_(H) and V_(L) regions that are each at least 95% identicalto the sequences shown in SEQ ID NO:68 and SEQ ID NO:69, respectively.

In an embodiment, a OX40 agonist comprises heavy chain CDR1, CDR2 andCDR3 domains having the sequences set forth in SEQ ID NO:70, SEQ IDNO:71, and SEQ ID NO:72, respectively, and conservative amino acidsubstitutions thereof, and light chain CDR1, CDR2 and CDR3 domainshaving the sequences set forth in SEQ ID NO:73, SEQ ID NO:74, and SEQ IDNO:75, respectively, and conservative amino acid substitutions thereof.

In an embodiment, the OX40 agonist is a OX40 agonist biosimilarmonoclonal antibody approved by drug regulatory authorities withreference to 11D4. In an embodiment, the biosimilar monoclonal antibodycomprises an OX40 antibody comprising an amino acid sequence which hasat least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequenceidentity, to the amino acid sequence of a reference medicinal product orreference biological product and which comprises one or morepost-translational modifications as compared to the reference medicinalproduct or reference biological product, wherein the reference medicinalproduct or reference biological product is 11D4. In some embodiments,the one or more post-translational modifications are selected from oneor more of: glycosylation, oxidation, deamidation, and truncation. Insome embodiments, the biosimilar is a OX40 agonist antibody authorizedor submitted for authorization, wherein the OX40 agonist antibody isprovided in a formulation which differs from the formulations of areference medicinal product or reference biological product, wherein thereference medicinal product or reference biological product is 11D4. TheOX40 agonist antibody may be authorized by a drug regulatory authoritysuch as the U.S. FDA and/or the European Union's EMA. In someembodiments, the biosimilar is provided as a composition which furthercomprises one or more excipients, wherein the one or more excipients arethe same or different to the excipients comprised in a referencemedicinal product or reference biological product, wherein the referencemedicinal product or reference biological product is 11D4. In someembodiments, the biosimilar is provided as a composition which furthercomprises one or more excipients, wherein the one or more excipients arethe same or different to the excipients comprised in a referencemedicinal product or reference biological product, wherein the referencemedicinal product or reference biological product is 11D4.

TABLE 11Amino acid sequences for OX40 agonist antibodies related to 11D4.Identifier Sequence (One-Letter Amino Acid Symbols) SEQ ID NO: 66EVQLVESGGG LVQPGGSLRL SCAASGFTFS SYSMNWVRQA PGKGLEWVSY ISSSSSTIDY  60heavy chain forADSVKGRFTI SRDNAKNSLY LQMNSLRDED TAVYYCARES GWYLFDYWGQ GTLVTVSSAS 12011D4TKGPSVFPLA PCSRSTSEST AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL 180YSLSSVVTVP SSNFGTQTYT CNVDHKPSNT KVDKTVERKC CVECPPCPAP PVAGPSVFLF 240PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV QFNWYVDGVE VHNAKTKPRE EQFNSTFRVV 300SVITVVHQDW LNGKEYKCKV SNKGLPAPIE KTISKTKGQP REPQVYTLPP SREEMTKNQV 360SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPMLDSDGS FFLYSKLTVD KSRWQQGNVF 420SCSVMHEALH NHYTQKSLSL SPGK                                        444SEQ ID NO: 67DIQMTQSPSS LSASVGDRVT ITCRASQGIS SWLAWYQQKP EKAPKSLIYA ASSLQSGVPS  60light chain forRFSGSGSGTD FTLTISSLQP EDFATYYCQQ YNSYPPTFGG GTKVEIKRTV AAPSVFIFPP 12011D4SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT 180LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC                             214SEQ ID NO: 68EVQLVESGGG LVQPGGSLRL SCAASGFTFS SYSMNWVRQA PGKGLEWVSY ISSSSSTIDY  60heavy chainADSVKGRFTI SRDNAKNSLY LQMNSLRDED TAVYYCARES GWYLFDYWGQ GTLVTVSS   118variable region for 11D4 SEQ ID NO: 69DIQMTQSPSS LSASVGDRVT ITCPASQGIS SWLAWYQQKP EKAPKSLIYA ASSLQSGVPS  60light chainRFSGSGSGTD FTLTISSLQP EDFATYYCQQ YNSYPPTFGG GTKVEIK               107variable region for 11D4 SEQ ID NO: 70SYSMN                                                               5heavy chain CDR1 for 11D4 SEQ ID NO: 71YISSSSSTID YADSVKG                                                 17heavy chain CDR2 for 11D4 SEQ ID NO: 72ESGWYLFDY                                                           9heavy chain CDR3 for 11D4 SEQ ID NO: 73RASQGISSWL A                                                       11light chain CDR1 for 11D4 SEQ ID NO: 74AASSLQS                                                             7light chain CDR2 for 11D4 SEQ ID NO: 75QQYNSYPPT                                                           9light chain CDR3 for 11D4

In some embodiments, the OX40 agonist is 18D8, which is a fully humanantibody available from Pfizer, Inc. The preparation and properties of18D8 are described in U.S. Pat. Nos. 7,960,515; 8,236,930; and9,028,824, the disclosures of which are incorporated by referenceherein. The amino acid sequences of 18D8 are set forth in Table 12.

In an embodiment, a OX40 agonist comprises a heavy chain given by SEQ IDNO:76 and a light chain given by SEQ ID NO:77. In an embodiment, a OX40agonist comprises heavy and light chains having the sequences shown inSEQ ID NO:76 and SEQ ID NO:77, respectively, or antigen bindingfragments, Fab fragments, single-chain variable fragments (scFv),variants, or conjugates thereof. In an embodiment, a OX40 agonistcomprises heavy and light chains that are each at least 99% identical tothe sequences shown in SEQ ID NO:76 and SEQ ID NO:77, respectively. Inan embodiment, a OX40 agonist comprises heavy and light chains that areeach at least 98% identical to the sequences shown in SEQ ID NO:76 andSEQ ID NO:77, respectively. In an embodiment, a OX40 agonist comprisesheavy and light chains that are each at least 97% identical to thesequences shown in SEQ ID NO:76 and SEQ ID NO:77, respectively. In anembodiment, a OX40 agonist comprises heavy and light chains that areeach at least 96% identical to the sequences shown in SEQ ID NO:76 andSEQ ID NO:77, respectively. In an embodiment, a OX40 agonist comprisesheavy and light chains that are each at least 95% identical to thesequences shown in SEQ ID NO:76 and SEQ ID NO:77, respectively.

In an embodiment, the OX40 agonist comprises the heavy and light chainCDRs or variable regions (VRs) of 18D8. In an embodiment, the OX40agonist heavy chain variable region (V_(H)) comprises the sequence shownin SEQ ID NO:78, and the OX40 agonist light chain variable region(V_(L)) comprises the sequence shown in SEQ ID NO:79, and conservativeamino acid substitutions thereof. In an embodiment, a OX40 agonistcomprises V_(H) and V_(L) regions that are each at least 99% identicalto the sequences shown in SEQ ID NO:78 and SEQ ID NO:79, respectively.In an embodiment, a OX40 agonist comprises V_(H) and V_(L) regions thatare each at least 98% identical to the sequences shown in SEQ ID NO:78and SEQ ID NO:79, respectively. In an embodiment, a OX40 agonistcomprises V_(H) and V_(L) regions that are each at least 97% identicalto the sequences shown in SEQ ID NO:78 and SEQ ID NO:79, respectively.In an embodiment, a OX40 agonist comprises V_(H) and V_(L) regions thatare each at least 96% identical to the sequences shown in SEQ ID NO:78and SEQ ID NO:79, respectively. In an embodiment, a OX40 agonistcomprises V_(H) and V_(L) regions that are each at least 95% identicalto the sequences shown in SEQ ID NO:78 and SEQ ID NO:79, respectively.

In an embodiment, a OX40 agonist comprises heavy chain CDR1, CDR2 andCDR3 domains having the sequences set forth in SEQ ID NO:80, SEQ IDNO:81, and SEQ ID NO:82, respectively, and conservative amino acidsubstitutions thereof, and light chain CDR1, CDR2 and CDR3 domainshaving the sequences set forth in SEQ ID NO:83, SEQ ID NO:84, and SEQ IDNO:85, respectively, and conservative amino acid substitutions thereof.

In an embodiment, the OX40 agonist is a OX40 agonist biosimilarmonoclonal antibody approved by drug regulatory authorities withreference to 18D8. In an embodiment, the biosimilar monoclonal antibodycomprises an OX40 antibody comprising an amino acid sequence which hasat least 97% sequence identity, e.g., 97%, 98%, 99% or 100% sequenceidentity, to the amino acid sequence of a reference medicinal product orreference biological product and which comprises one or morepost-translational modifications as compared to the reference medicinalproduct or reference biological product, wherein the reference medicinalproduct or reference biological product is 18D8. In some embodiments,the one or more post-translational modifications are selected from oneor more of: glycosylation, oxidation, deamidation, and truncation. Insome embodiments, the biosimilar is a OX40 agonist antibody authorizedor submitted for authorization, wherein the OX40 agonist antibody isprovided in a formulation which differs from the formulations of areference medicinal product or reference biological product, wherein thereference medicinal product or reference biological product is 18D8. TheOX40 agonist antibody may be authorized by a drug regulatory authoritysuch as the U.S. FDA and/or the European Union's EMA. In someembodiments, the biosimilar is provided as a composition which furthercomprises one or more excipients, wherein the one or more excipients arethe same or different to the excipients comprised in a referencemedicinal product or reference biological product, wherein the referencemedicinal product or reference biological product is 18D8. In someembodiments, the biosimilar is provided as a composition which furthercomprises one or more excipients, wherein the one or more excipients arethe same or different to the excipients comprised in a referencemedicinal product or reference biological product, wherein the referencemedicinal product or reference biological product is 18D8.

TABLE 12Amino acid sequences for OX40 agonist antibodies related to 18D8.Identifier Sequence (One-Letter Amino Acid Symbols) SEQ ID NO: 76EVQLVESGGG LVQPGRSLRL SCAASGFTFD DYAMHWVRQA PGKGLEWVSG ISWNSGSIGY  60heavy chain forADSVKGRFTI SRDNAKNSLY LQMNSLRAED TALYYCAKDQ STADYYFYYG MDVWGQGTTV 12018D8TVSSASTKGP SVFPLAPCSR STSESTAALG CLVKDYFPEP VTVSWNSGAL TSGVHTFPAV 180LQSSGLYSLS SVVTVPSSNF GTQTYTCNVD HKPSNTKVDK TVERKCCVEC PPCPAPPVAG 240PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVQFNW YVDGVEVHNA KTKPREEQFN 300STERVVSVIT VVHQDWLNGK EYKCKVSNKG LPAPIEKTIS KTKGQPREPQ VYTLPPSREE 360MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPM LDSDGSFFLY SKLTVDKSRW 420QQGNVFSCSV MHEALHNHYT QKSLSLSPGK                                  450SEQ ID NO: 77EIVVTQSPAT LSLSPGERAT LSCPASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA  60light chain forRFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPTFGQG TKVEIKRTVA APSVFIFPPS 12018D8DEQLKSGTAS VVCLLNNFYP REAKVQWKVD NALQSGNSQE SVTEQDSKDS TYSLSSTLTL 180SKADYEKHKV YACEVTHQGL SSPVTKSFNR GEC                              213SEQ ID NO: 78EVQLVESGGG LVQPGRSLRL SCAASGFTFD DYAMHWVRQA PGKGLEWVSG ISWNSGSIGY  60heavy chainADSVKGRFTI SRDNAKNSLY LQMNSLRAED TALYYCAKDQ STADYYFYYG MDVWGQGTTV 120variable regionTVSS                                                              124for 18D8 SEQ ID NO: 79EIVVTQSPAT LSLSPGERAT LSCPASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA  60light chainRFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPTFGQG TKVEIK                106variable region for 18D8 SEQ ID NO: 80DYAMH                                                               5heavy chain CDR1 for 18D8 SEQ ID NO: 81GISWNSGSIG YADSVKG                                                 17heavy chain CDR2 for 18D8 SEQ ID NO: 82DQSTADYYFY YGMDV                                                   15heavy chain CDR3 for 18D8 SEQ ID NO: 83RASQSVSSYL A                                                       11light chain CDR1 for 18D8 SEQ ID NO: 84DASNRAT                                                             7light chain CDR2 for 18D8 SEQ ID NO: 85QQRSNWPT                                                            8light chain CDR3 for 18D8

In some embodiments, the OX40 agonist is Hu119-122, which is a humanizedantibody available from GlaxoSmithKline plc. The preparation andproperties of Hu119-122 are described in U.S. Pat. Nos. 9,006,399 and9,163,085, and in International Patent Publication No. WO 2012/027328,the disclosures of which are incorporated by reference herein. The aminoacid sequences of Hu119-122 are set forth in Table 13.

In an embodiment, the OX40 agonist comprises the heavy and light chainCDRs or variable regions (VRs) of Hu119-122. In an embodiment, the OX40agonist heavy chain variable region (V_(H)) comprises the sequence shownin SEQ ID NO:86, and the OX40 agonist light chain variable region(V_(L)) comprises the sequence shown in SEQ ID NO:87, and conservativeamino acid substitutions thereof. In an embodiment, a OX40 agonistcomprises V_(H) and V_(L) regions that are each at least 99% identicalto the sequences shown in SEQ ID NO:86 and SEQ ID NO:87, respectively.In an embodiment, a OX40 agonist comprises V_(H) and V_(L) regions thatare each at least 98% identical to the sequences shown in SEQ ID NO:86and SEQ ID NO:87, respectively. In an embodiment, a OX40 agonistcomprises V_(H) and V_(L) regions that are each at least 97% identicalto the sequences shown in SEQ ID NO:86 and SEQ ID NO:87, respectively.In an embodiment, a OX40 agonist comprises V_(H) and V_(L) regions thatare each at least 96% identical to the sequences shown in SEQ ID NO:86and SEQ ID NO:87, respectively. In an embodiment, a OX40 agonistcomprises V_(H) and V_(L) regions that are each at least 95% identicalto the sequences shown in SEQ ID NO:86 and SEQ ID NO:87, respectively.

In an embodiment, a OX40 agonist comprises heavy chain CDR1, CDR2 andCDR3 domains having the sequences set forth in SEQ ID NO:88, SEQ IDNO:89, and SEQ ID NO:90, respectively, and conservative amino acidsubstitutions thereof, and light chain CDR1, CDR2 and CDR3 domainshaving the sequences set forth in SEQ ID NO:91, SEQ ID NO:92, and SEQ IDNO:93, respectively, and conservative amino acid substitutions thereof.

In an embodiment, the OX40 agonist is a OX40 agonist biosimilarmonoclonal antibody approved by drug regulatory authorities withreference to Hu119-122. In an embodiment, the biosimilar monoclonalantibody comprises an OX40 antibody comprising an amino acid sequencewhich has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100%sequence identity, to the amino acid sequence of a reference medicinalproduct or reference biological product and which comprises one or morepost-translational modifications as compared to the reference medicinalproduct or reference biological product, wherein the reference medicinalproduct or reference biological product is Hu119-122. In someembodiments, the one or more post-translational modifications areselected from one or more of glycosylation, oxidation, deamidation, andtruncation. In some embodiments, the biosimilar is a OX40 agonistantibody authorized or submitted for authorization, wherein the OX40agonist antibody is provided in a formulation which differs from theformulations of a reference medicinal product or reference biologicalproduct, wherein the reference medicinal product or reference biologicalproduct is Hu119-122. The OX40 agonist antibody may be authorized by adrug regulatory authority such as the U.S. FDA and/or the EuropeanUnion's EMA. In some embodiments, the biosimilar is provided as acomposition which further comprises one or more excipients, wherein theone or more excipients are the same or different to the excipientscomprised in a reference medicinal product or reference biologicalproduct, wherein the reference medicinal product or reference biologicalproduct is Hu119-122. In some embodiments, the biosimilar is provided asa composition which further comprises one or more excipients, whereinthe one or more excipients are the same or different to the excipientscomprised in a reference medicinal product or reference biologicalproduct, wherein the reference medicinal product or reference biologicalproduct is Hu119-122.

TABLE 13Amino acid sequences for OX40 agonist antibodies related to Hu119-122.Identifier Sequence (One-Letter Amino Acid Symbols) SEQ ID NO: 86EVQLVESGGG LVQPGGSLRL SCAASEYEFP SHDMSWVRQA PGKGLELVAA INSDGGSTYY  60heavy chainPDTMERRFTI SRDNAKNSLY LQMNSLRAED TAVYYCARHY DDYYAWFAYW GQGTMVTVSS 120variable region for Hu119-122 SEQ ID NO: 87EIVLTQSPAT LSLSPGERAT LSCPASKSVS TSGYSYMHWY QQKPGQAPRL LIYLASNLES  60light chainGVPARFSGSG SGTDFTLTIS SLEPEDFAVY YCQHSRELPL TFGGGTKVEI K          111variable region for Hu119-122 SEQ ID NO: 88SHDMS                                                               5heavy chain CDR1 for Hu119-122 SEQ ID NO: 89AINSDGGSTY YPDTMER                                                 17heavy chain CDR2 for Hu119-122 SEQ ID NO: 90HYDDYYAWFA Y                                                       11heavy chain CDR3 for Hu119-122 SEQ ID NO: 91RASKSVSTSG YSYMH                                                   15light chain CDR1 for Hu119-122 SEQ ID NO: 92LASNLES                                                             7light chain CDR2 for Hu119-122 SEQ ID NO: 93QHSRELPLT                                                           9light chain CDR3 for Hu119-122

In some embodiments, the OX40 agonist is Hu106-222, which is a humanizedantibody available from GlaxoSmithKline plc. The preparation andproperties of Hu106-222 are described in U.S. Pat. Nos. 9,006,399 and9,163,085, and in International Patent Publication No. WO 2012/027328,the disclosures of which are incorporated by reference herein. The aminoacid sequences of Hu106-222 are set forth in Table 14.

In an embodiment, the OX40 agonist comprises the heavy and light chainCDRs or variable regions (VRs) of Hu106-222. In an embodiment, the OX40agonist heavy chain variable region (V_(H)) comprises the sequence shownin SEQ ID NO:94, and the OX40 agonist light chain variable region(V_(L)) comprises the sequence shown in SEQ ID NO:95, and conservativeamino acid substitutions thereof. In an embodiment, a OX40 agonistcomprises V_(H) and V_(L) regions that are each at least 99% identicalto the sequences shown in SEQ ID NO:94 and SEQ ID NO:95, respectively.In an embodiment, a OX40 agonist comprises V_(H) and V_(L) regions thatare each at least 98% identical to the sequences shown in SEQ ID NO:94and SEQ ID NO:95, respectively. In an embodiment, a OX40 agonistcomprises V_(H) and V_(L) regions that are each at least 97% identicalto the sequences shown in SEQ ID NO:94 and SEQ ID NO:95, respectively.In an embodiment, a OX40 agonist comprises V_(H) and V_(L) regions thatare each at least 96% identical to the sequences shown in SEQ ID NO:94and SEQ ID NO:95, respectively. In an embodiment, a OX40 agonistcomprises V_(H) and V_(L) regions that are each at least 95% identicalto the sequences shown in SEQ ID NO:94 and SEQ ID NO:95, respectively.

In an embodiment, a OX40 agonist comprises heavy chain CDR1, CDR2 andCDR3 domains having the sequences set forth in SEQ ID NO:96, SEQ IDNO:97, and SEQ ID NO:98, respectively, and conservative amino acidsubstitutions thereof, and light chain CDR1, CDR2 and CDR3 domainshaving the sequences set forth in SEQ ID NO:99, SEQ ID NO:100, and SEQID NO:101, respectively, and conservative amino acid substitutionsthereof.

In an embodiment, the OX40 agonist is a OX40 agonist biosimilarmonoclonal antibody approved by drug regulatory authorities withreference to Hu106-222. In an embodiment, the biosimilar monoclonalantibody comprises an OX40 antibody comprising an amino acid sequencewhich has at least 97% sequence identity, e.g., 97%, 98%, 99% or 100%sequence identity, to the amino acid sequence of a reference medicinalproduct or reference biological product and which comprises one or morepost-translational modifications as compared to the reference medicinalproduct or reference biological product, wherein the reference medicinalproduct or reference biological product is Hu106-222. In someembodiments, the one or more post-translational modifications areselected from one or more of: glycosylation, oxidation, deamidation, andtruncation. In some embodiments, the biosimilar is a OX40 agonistantibody authorized or submitted for authorization, wherein the OX40agonist antibody is provided in a formulation which differs from theformulations of a reference medicinal product or reference biologicalproduct, wherein the reference medicinal product or reference biologicalproduct is Hu106-222. The OX40 agonist antibody may be authorized by adrug regulatory authority such as the U.S. FDA and/or the EuropeanUnion's EMA. In some embodiments, the biosimilar is provided as acomposition which further comprises one or more excipients, wherein theone or more excipients are the same or different to the excipientscomprised in a reference medicinal product or reference biologicalproduct, wherein the reference medicinal product or reference biologicalproduct is Hu106-222. In some embodiments, the biosimilar is provided asa composition which further comprises one or more excipients, whereinthe one or more excipients are the same or different to the excipientscomprised in a reference medicinal product or reference biologicalproduct, wherein the reference medicinal product or reference biologicalproduct is Hu106-222.

TABLE 14Amino acid sequences for OX40 agonist antibodies related to Hu106-222.Identifier Sequence (One-Letter Amino Acid Symbols) SEQ ID NO: 94QVQLVQSGSE LKKPGASVKV SCKASGYTFT DYSMHWVRQA PGQGLKWMGW INTETGEPTY  60heavy chainADDFKGRFVF SLDTSVSTAY LQISSLKAED TAVYYCANPY YDYVSYYAMD YWGQGTTVTV 120variable regionSS                                                                122for Hu106-222 SEQ ID NO: 95DIQMTQSPSS LSASVGDRVT ITCKASQDVS TAVAWYQQKP GKAPKILIYS ASYLYTGVPS  60light chainRFSGSGSGTD FTFTISSLQP EDIATYYCQQ HYSTPRTFGQ GTKLEIK               107variable region for Hu106-222 SEQ ID NO: 96DYSMH                                                               5heavy chain CDR1 for Hu106-222 SEQ ID NO: 97WINTETGEPT YADDFKG                                                 17heavy chain CDR2 for Hu106-222 SEQ ID NO: 98PYYDYVSYYA MDY                                                     13heavy chain CDR3 for Hu106-222 SEQ ID NO: 99KASQDVSTAV A                                                       11light chain CDR1 for Hu106-222 SEQ ID NO: 100SASYLYT                                                             7light chain CDR2 for Hu106-222 SEQ ID NO: 101QQHYSTPRT                                                           9light chain CDR3 for Hu106-222

In some embodiments, the OX40 agonist antibody is MEDI6469 (alsoreferred to as 9B12). MEDI6469 is a murine monoclonal antibody.Weinberg, et al., J. Immunother. 2006, 29, 575-585. In some embodimentsthe OX40 agonist is an antibody produced by the 9B12 hybridoma,deposited with Biovest Inc. (Malvern, Mass., USA), as described inWeinberg, et al., J. Immunother. 2006, 29, 575-585, the disclosure ofwhich is hereby incorporated by reference in its entirety. In someembodiments, the antibody comprises the CDR sequences of MEDI6469. Insome embodiments, the antibody comprises a heavy chain variable regionsequence and/or a light chain variable region sequence of MEDI6469.

In an embodiment, the OX40 agonist is L106 BD (Pharmingen Product#340420). In some embodiments, the OX40 agonist comprises the CDRs ofantibody L106 (BD Pharmingen Product #340420). In some embodiments, theOX40 agonist comprises a heavy chain variable region sequence and/or alight chain variable region sequence of antibody L106 (BD PharmingenProduct #340420). In an embodiment, the OX40 agonist is ACT35 (SantaCruz Biotechnology, Catalog #20073). In some embodiments, the OX40agonist comprises the CDRs of antibody ACT35 (Santa Cruz Biotechnology,Catalog #20073). In some embodiments, the OX40 agonist comprises a heavychain variable region sequence and/or a light chain variable regionsequence of antibody ACT35 (Santa Cruz Biotechnology, Catalog #20073).In an embodiment, the OX40 agonist is the murine monoclonal antibodyanti-mCD134/mOX40 (clone OX86), commercially available from InVivoMAb,BioXcell Inc, West Lebanon, N.H.

In an embodiment, the OX40 agonist is selected from the OX40 agonistsdescribed in International Patent Application Publication Nos. WO95/12673, WO 95/21925, WO 2006/121810, WO 2012/027328, WO 2013/028231,WO 2013/038191, and WO 2014/148895; European Patent Application EP0672141; U.S. Patent Application Publication Nos. US 2010/136030, US2014/377284, US 2015/190506, and US 2015/132288 (including clones 20E5and 12H3); and U.S. Pat. Nos. 7,504,101, 7,550,140, 7,622,444,7,696,175, 7,960,515, 7,961,515, 8,133,983, 9,006,399, and 9,163,085,the disclosure of each of which is incorporated herein by reference inits entirety.

In an embodiment, the OX40 agonist is an OX40 agonistic fusion proteinas depicted in Structure I-A (C-terminal Fc-antibody fragment fusionprotein) or Structure I-B (N-terminal Fc-antibody fragment fusionprotein), or a fragment, derivative, conjugate, variant, or biosimilarthereof. The properties of structures I-A and I-B are described aboveand in U.S. Pat. Nos. 9,359,420, 9,340,599, 8,921,519, and 8,450,460,the disclosures of which are incorporated by reference herein. Aminoacid sequences for the polypeptide domains of structure I-A are given inTable 6. The Fc domain preferably comprises a complete constant domain(amino acids 17-230 of SEQ ID NO:31) the complete hinge domain (aminoacids 1-16 of SEQ ID NO:31) or a portion of the hinge domain (e.g.,amino acids 4-16 of SEQ ID NO:31). Preferred linkers for connecting aC-terminal Fc-antibody may be selected from the embodiments given in SEQID NO:32 to SEQ ID NO:41, including linkers suitable for fusion ofadditional polypeptides. Likewise, amino acid sequences for thepolypeptide domains of structure I-B are given in Table 7. If an Fcantibody fragment is fused to the N-terminus of an TNRFSF fusion proteinas in structure I-B, the sequence of the Fc module is preferably thatshown in SEQ ID NO:42, and the linker sequences are preferably selectedfrom those embodiments set forth in SEQ ID NO:43 to SEQ ID NO:45.

In an embodiment, an OX40 agonist fusion protein according to structuresI-A or I-B comprises one or more OX40 binding domains selected from thegroup consisting of a variable heavy chain and variable light chain oftavolixizumab, a variable heavy chain and variable light chain of 11D4,a variable heavy chain and variable light chain of 18D8, a variableheavy chain and variable light chain of Hu119-122, a variable heavychain and variable light chain of Hu106-222, a variable heavy chain andvariable light chain selected from the variable heavy chains andvariable light chains described in Table 15, any combination of avariable heavy chain and variable light chain of the foregoing, andfragments, derivatives, conjugates, variants, and biosimilars thereof.

In an embodiment, an OX40 agonist fusion protein according to structuresI-A or I-B comprises one or more OX40 binding domains comprising anOX40L sequence. In an embodiment, an OX40 agonist fusion proteinaccording to structures I-A or I-B comprises one or more OX40 bindingdomains comprising a sequence according to SEQ ID NO:102. In anembodiment, an OX40 agonist fusion protein according to structures I-Aor I-B comprises one or more OX40 binding domains comprising a solubleOX40L sequence. In an embodiment, a OX40 agonist fusion proteinaccording to structures I-A or I-B comprises one or more OX40 bindingdomains comprising a sequence according to SEQ ID NO:103. In anembodiment, a OX40 agonist fusion protein according to structures I-A orI-B comprises one or more OX40 binding domains comprising a sequenceaccording to SEQ ID NO:104.

In an embodiment, an OX40 agonist fusion protein according to structuresI-A or I-B comprises one or more OX40 binding domains that is a scFvdomain comprising V_(H) and V_(L) regions that are each at least 95%identical to the sequences shown in SEQ ID NO:58 and SEQ ID NO:59,respectively, wherein the V_(H) and V_(L) domains are connected by alinker. In an embodiment, an OX40 agonist fusion protein according tostructures I-A or I-B comprises one or more OX40 binding domains that isa scFv domain comprising V_(H) and V_(L) regions that are each at least95% identical to the sequences shown in SEQ ID NO:68 and SEQ ID NO:69,respectively, wherein the V_(H) and V_(L) domains are connected by alinker. In an embodiment, an OX40 agonist fusion protein according tostructures I-A or I-B comprises one or more OX40 binding domains that isa scFv domain comprising V_(H) and V_(L) regions that are each at least95% identical to the sequences shown in SEQ ID NO:78 and SEQ ID NO:79,respectively, wherein the V_(H) and V_(L) domains are connected by alinker. In an embodiment, an OX40 agonist fusion protein according tostructures I-A or I-B comprises one or more OX40 binding domains that isa scFv domain comprising V_(H) and V_(L) regions that are each at least95% identical to the sequences shown in SEQ ID NO:86 and SEQ ID NO:87,respectively, wherein the V_(H) and V_(L) domains are connected by alinker. In an embodiment, an OX40 agonist fusion protein according tostructures I-A or I-B comprises one or more OX40 binding domains that isa scFv domain comprising V_(H) and V_(L) regions that are each at least95% identical to the sequences shown in SEQ ID NO:94 and SEQ ID NO:95,respectively, wherein the V_(H) and V_(L) domains are connected by alinker. In an embodiment, an OX40 agonist fusion protein according tostructures I-A or I-B comprises one or more OX40 binding domains that isa scFv domain comprising V_(H) and V_(L) regions that are each at least95% identical to the V_(H) and V_(L) sequences given in Table 15,wherein the V_(H) and V_(L) domains are connected by a linker.

TABLE 15Additional polypeptide domains useful as OX40 binding domains in fusionproteins (e.g., structures I-A and I-B) or as scFv OX40 agonist antibodies.Identifier Sequence (One-Letter Amino Acid Symbols) SEQ ID NO: 102MERVQPLEEN VGNAARPRFE RNKLLLVASV IQGLGLLLCF TYICLHFSAL QVSHRYPRIQ  60OX40LSIKVQFTEYK KEKGFILTSQ KEDEIMKVQN NSVIINCDGF YLISLKGYFS QEVNISLHYQ 120KDEEPLFQLK KVRSVNSLMV ASLTYKDKVY LNVTTDNTSL DDFHVNGGEL ILIHQNPGEF 180CVL                                                               183SEQ ID NO: 103SHRYPRIQSI KVQFTEYKKE KGFILTSQKE DEIMKVQNNS VIINCDGFYL ISLKGYFSQE  60OX4OL solubleVNISLHYQKD EEPLFQLKKV RSVNSLMVAS LTYKDKVYLN VTTDNTSLDD FHVNGGELIL 120domainIHQNPGEFCV L                                                      131SEQ ID NO: 104YPRIQSIKVQ FTEYKKEKGF ILTSQKEDEI MKVQNNSVII NCDGFYLISL KGYFSQEVNI  60OX4OL solubleSLHYQKDEEP LFQLKKVRSV NSLMVASLTY KDKVYLNVTT DNTSLDDFHV NGGELILIHQ 120domainNPGEFCVL                                                          128(alternative) SEQ ID NO: 105EVQLVESGGG LVQPGGSLRL SCAASGFTFS NYTMNWVRQA PGKGLEWVSA ISGSGGSTYY  60variable heavyADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKDR YSQVHYALDY WGQGTLVTVS 120chain for 008 SEQ ID NO: 106DIVMTQSPDS LPVTPGEPAS ISCRSSQSLL HSNGYNYLDW YLQKAGQSPQ LLIYLGSNRA  60variable lightSGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCQQYYNHP TTFGQGTK              108chain for 008 SEQ ID NO: 107EVQLVESGGG VVQPGRSLRL SCAASGFTFS DYTMNWVRQA PGKGLEWVSS ISGGSTYYAD  60variable heavySRKGRFTISR DNSKNTLYLQ MNNLRAEDTA VYYCARDRYF RQQNAFDYWG QGTLVTVSSA 120chain for 011 SEQ ID NO: 108DIVMTQSPDS LPVTPGEPAS ISCRSSQSLL HSNGYNYLDW YLQKAGQSPQ LLIYLGSNRA  60variable lightSGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCQQYYNHP TTFGQGTK              108chain for 011 SEQ ID NO: 109EVQLVESGGG LVQPRGSLRL SCAASGFTFS SYAMNWVRQA PGKGLEWVAV ISYDGSNKYY  variable heavyADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKDR YITLPNALDY WGQGTLVTVS 120chain for 021 SEQ ID NO: 110DIQMTQSPVS LPVTPGEPAS ISCRSSQSLL HSNGYNYLDW YLQKPGQSPQ LLIYLGSNRA  60variable lightSGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCQQYKSNP PTFGQGTK              108chain for 021 SEQ ID NO: 111EVQLVESGGG LVHPGGSLRL SCAGSGFTFS SYAMHWVRQA PGKGLEWVSA IGTGGGTYYA  60variable heavyDSVMGRFTIS RDNSKNTLYL QMNSLRAEDT AVYYCARYDN VMGLYWFDYW GQGTLVTVSS 120chain for 023 SEQ ID NO: 112EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA  60variable lightRFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPPAFGG GTKVEIKR              108chain for 023 SEQ ID NO: 113EVQLQQSGPE LVKPGASVKM SCKASGYTFT SYVMHWVKQK PGQGLEWIGY INPYNDGTKY  60heavy chainNEKFKGKATL TSDKSSSTAY MELSSLTSED SAVYYCANYY GSSLSMDYWG QGTSVTVSS  119variable region SEQ ID NO: 114DIQMTQTTSS LSASLGDRVT ISCRASQDIS NYLNWYQQKP DGTVKLLIYY TSRLHSGVPS  60light chainRFSGSGSGTD YSLTISNLEQ EDIATYFCQQ GNTLPWTFGG GTKLEIKR              108variable region SEQ ID NO: 115EVQLQQSGPE LVKPGASVKI SCKTSGYTFK DYTMHWVKQS HGKSLEWIGG IYPNNGGSTY  60heavy chainNQNFKDKATL TVDKSSSTAY MEFRSLTSED SAVYYCARMG YHGPHLDFDV WGAGTTVTVS 120variable regionP                                                                 121SEQ ID NO: 116DIVMTQSHKF MSTSLGDRVS ITCKASQDVG AAVAWYQQKP GQSPKLLIYW ASTRHTGVPD  60light chainRFTGGGSGTD FTLTISNVQS EDLTDYFCQQ YINYPLTFGG GTKLEIKR              108variable region SEQ ID NO: 117QIQLVQSGPE LKKPGETVKI SCKASGYTFT DYSMHWVKQA PGKGLKWMGW INTETGEPTY  60heavy chainADDFKGRFAF SLETSASTAY LQINNLKNED TATYFCANPY YDYVSYYAMD YWGHGTSVTV 120variable regionSS                                                                122of humanized antibody SEQ ID NO: 118QVQLVQSGSE LKKPGASVKV SCKASGYTFT DYSMHWVRQA PGQGLKWMGW INTETGEPTY  60heavy chainADDFKGRFVF SLDTSVSTAY LQISSLKAED TAVYYCANPY YDYVSYYAMD YWGQGTTVTV 120variable regionSS                                                                122of humanized antibody SEQ ID NO: 119DIVMTQSHKF MSTSVRDRVS ITCKASQDVS TAVAWYQQKP GQSPKLLIYS ASYLYTGVPD  60light chainRFTGSGSGTD FTFTISSVQA EDLAVYYCQQ HYSTPRTFGG GTKLEIK               107variable region of humanized antibody SEQ ID NO: 120DIVMTQSHKF MSTSVRDRVS ITCKASQDVS TAVAWYQQKP GQSPKLLIYS ASYLYTGVPD  60light chainRFTGSGSGTD FTFTISSVQA EDLAVYYCQQ HYSTPRTFGG GTKLEIK               107variable region of humanized antibody SEQ ID NO: 121EVQLVESGGG LVQPGESLKL SCESNEYEFP SHDMSWVRKT PEKRLELVAA INSDGGSTYY  60heavy chainPDTMERRFII SRDNTKKTLY LQMSSLRSED TALYYCARHY DDYYAWFAYW GQGTLVTVSA 120variable region of humanized antibody SEQ ID NO: 122EVQLVESGGG LVQPGGSLRL SCAASEYEFP SHDMSWVRQA PGKGLELVAA INSDGGSTYY  60heavy chainPDTMERRFTI SRDNAKNSLY LQMNSLRAED TAVYYCARHY DDYYAWFAYW GQGTMVTVSS 120variable region of humanized antibody SEQ ID NO: 123DIVLTQSPAS LAVSLGQRAT ISCRASKSVS TSGYSYMHWY QQKPGQPPKL LIYLASNLES  60light chainGVPARFSGSG SGTDFTLNIH PVEEEDAATY YCQHSRELPL TFGAGTKLEL K          111variable region of humanized antibody SEQ ID NO: 124EIVLTQSPAT LSLSPGERAT LSCRASKSVS TSGYSYMHWY QQKPGQAPRL LIYLASNLES  60light chainGVPARFSGSG SGTDFTLTIS SLEPEDFAVY YCQHSRELPL TFGGGTKVEI K          111variable region of humanized antibody SEQ ID NO: 125MYLGLNYVFI VFLLNGVQSE VKLEESGGGL VQPGGSMKLS CAASGFTFSD AWMDWVRQSP  60heavy chainEKGLEWVAEI RSKANNHATY YAESVNGRFT ISRDDSKSSV YLQMNSLRAE DTGIYYCTWG 120variable regionEVFYFDYWGQ GTTLTVSS                                               138SEQ ID NO: 126MRPSIQFLGL LLFWLHGAQC DIQMTQSPSS LSASLGGKVT ITCKSSQDIN KYIAWYQHKP  60light chainGKGPRLLIHY TSTLQPGIPS RFSGSGSGRD YSFSISNLEP EDIATYYCLQ YDNLLTFGAG 120variable regionTKLELK                                                            126

In an embodiment, the OX40 agonist is a OX40 agonistic single-chainfusion polypeptide comprising (i) a first soluble OX40 binding domain,(ii) a first peptide linker, (iii) a second soluble OX40 binding domain,(iv) a second peptide linker, and (v) a third soluble OX40 bindingdomain, further comprising an additional domain at the N-terminal and/orC-terminal end, and wherein the additional domain is a Fab or Fcfragment domain. In an embodiment, the OX40 agonist is a OX40 agonisticsingle-chain fusion polypeptide comprising (i) a first soluble OX40binding domain, (ii) a first peptide linker, (iii) a second soluble OX40binding domain, (iv) a second peptide linker, and (v) a third solubleOX40 binding domain, further comprising an additional domain at theN-terminal and/or C-terminal end, wherein the additional domain is a Fabor Fc fragment domain wherein each of the soluble OX40 binding domainslacks a stalk region (which contributes to trimerisation and provides acertain distance to the cell membrane, but is not part of the OX40binding domain) and the first and the second peptide linkersindependently have a length of 3-8 amino acids.

In an embodiment, the OX40 agonist is an OX40 agonistic single-chainfusion polypeptide comprising (i) a first soluble tumor necrosis factor(TNF) superfamily cytokine domain, (ii) a first peptide linker, (iii) asecond soluble TNF superfamily cytokine domain, (iv) a second peptidelinker, and (v) a third soluble TNF superfamily cytokine domain, whereineach of the soluble TNF superfamily cytokine domains lacks a stalkregion and the first and the second peptide linkers independently have alength of 3-8 amino acids, and wherein the TNF superfamily cytokinedomain is an OX40 binding domain.

In some embodiments, the OX40 agonist is MEDI6383. MEDI6383 is an OX40agonistic fusion protein and can be prepared as described in U.S. Pat.No. 6,312,700, the disclosure of which is incorporated by referenceherein.

In an embodiment, the OX40 agonist is an OX40 agonistic scFv antibodycomprising any of the foregoing V_(H) domains linked to any of theforegoing V_(L) domains.

In an embodiment, the OX40 agonist is Creative Biolabs OX40 agonistmonoclonal antibody MOM-18455, commercially available from CreativeBiolabs, Inc., Shirley, N.Y., USA.

In an embodiment, the OX40 agonist is OX40 agonistic antibody cloneBer-ACT35 commercially available from BioLegend, Inc., San Diego,Calif., USA.

E. STEP E: Harvest TILS

After the second expansion step, cells can be harvested. In someembodiments the TILs are harvested after one, two, three, four or moreexpansion steps, for example as provided in FIG. 9. In some embodimentsthe TILs are harvested after two expansion steps, for example asprovided in FIG. 9.

TILs can be harvested in any appropriate and sterile manner, includingfor example by centrifugation. Methods for TIL harvesting are well knownin the art and any such know methods can be employed with the presentprocess. In some embodiments, TILS are harvest using an automatedsystem.

Cell harvesters and/or cell processing systems are commerciallyavailable from a variety of sources, including, for example, FreseniusKabi, Tomtec Life Science, Perkin Elmer, and Inotech BiosystemsInternational, Inc. Any cell based harvester can be employed with thepresent methods. In some embodiments, the cell harvester and/or cellprocessing systems is a membrane-based cell harvester. In someembodiments, cell harvesting is via a cell processing system, such asthe LOVO system (manufactured by Fresenius Kabi). The term “LOVO cellprocessing system” also refers to any instrument or device manufacturedby any vendor that can pump a solution comprising cells through amembrane or filter such as a spinning membrane or spinning filter in asterile and/or closed system environment, allowing for continuous flowand cell processing to remove supernatant or cell culture media withoutpelletization. In some embodiments, the cell harvester and/or cellprocessing system can perform cell separation, washing, fluid-exchange,concentration, and/or other cell processing steps in a closed, sterilesystem.

In some embodiments, the harvest, for example, Step E according to FIG.9, is performed from a closed system bioreactor. In some embodiments, aclosed system is employed for the TIL expansion, as described herein. Insome embodiments, a single bioreactor is employed. In some embodiments,the single bioreactor employed is for example a G-REX-10 or a G-REX-100.In some embodiments, the closed system bioreactor is a singlebioreactor.

F. STEP F: Final Formulation and Transfer to Infusion Bag

After Steps A through E as provided in an exemplary order in FIG. 9 andas outlined in detailed above and herein are complete, cells aretransferred to a container for use in administration to a patient. Insome embodiments, once a therapeutically sufficient number of TILs areobtained using the expansion methods described above, they aretransferred to a container for use in administration to a patient.

In an embodiment, TILs expanded using APCs of the present disclosure areadministered to a patient as a pharmaceutical composition. In anembodiment, the pharmaceutical composition is a suspension of TILs in asterile buffer. TILs expanded using PBMCs of the present disclosure maybe administered by any suitable route as known in the art. In someembodiments, the T-cells are administered as a single intra-arterial orintravenous infusion, which preferably lasts approximately 30 to 60minutes. Other suitable routes of administration includeintraperitoneal, intrathecal, and intralymphatic.

G. Pharmaceutical Compositions, Dosages, and Dosing Regimens

In an embodiment, TILs expanded using the methods of the presentdisclosure are administered to a patient as a pharmaceuticalcomposition. In an embodiment, the pharmaceutical composition is asuspension of TILs in a sterile buffer. TILs expanded using PBMCs of thepresent disclosure may be administered by any suitable route as known inthe art. In some embodiments, the T-cells are administered as a singleintra-arterial or intravenous infusion, which preferably lastsapproximately 30 to 60 minutes. Other suitable routes of administrationinclude intraperitoneal, intrathecal, and intralymphatic administration.

Any suitable dose of TILs can be administered. In some embodiments, fromabout 2.3×10¹⁰ to about 13.7×10¹⁰ TILs are administered, with an averageof around 7.8×10¹⁰ TILs, particularly if the cancer is melanoma. In anembodiment, about 1.2×10¹⁰ to about 4.3×10¹⁰ of TILs are administered.In some embodiments, about 3×10¹⁰ to about 12×10¹⁰ TILs areadministered. In some embodiments, about 4×10¹⁰ to about 10×10¹⁰ TILsare administered. In some embodiments, about 5×10¹⁰ to about 8×10¹⁰ TILsare administered. In some embodiments, about 6×10¹⁰ to about 8×10¹⁰ TILsare administered. In some embodiments, about 7×10¹⁰ to about 8×10¹⁰ TILsare administered. In some embodiments, the therapeutically effectivedosage is about 2.3×10¹⁰ to about 13.7×10¹⁰. In some embodiments, thetherapeutically effective dosage is about 7.8×10¹⁰ TILs, particularly ofthe cancer is melanoma. In some embodiments, the therapeuticallyeffective dosage is about 1.2×10¹⁰ to about 4.3×10¹⁰ of TILs. In someembodiments, the therapeutically effective dosage is about 3×10¹⁰ toabout 12×10¹⁰ TILs. In some embodiments, the therapeutically effectivedosage is about 4×10¹⁰ to about 10×10¹⁰ TILs. In some embodiments, thetherapeutically effective dosage is about 5×10¹⁰ to about 8×10¹⁰ TILs.In some embodiments, the therapeutically effective dosage is about6×10¹⁰ to about 8×10¹⁰ TILs. In some embodiments, the therapeuticallyeffective dosage is about 7×10¹⁰ to about 8×10¹⁰ TILs.

In some embodiments, the number of the TILs provided in thepharmaceutical compositions of the invention is about 1×10⁶, 2×10⁶,3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷,4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸,5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹,6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰,6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹,6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, 1×10¹², 2×10¹², 3×10¹², 4×10¹², 5×10¹²,6×10¹², 7×10¹², 8×10², 9×10¹², 1×10¹³, 2×10¹³, 3×10¹³, 4×10¹³, 5×10¹³,6×10¹³, 7×10¹³, 8×10¹³, and 9×10¹³. In an embodiment, the number of theTILs provided in the pharmaceutical compositions of the invention is inthe range of 1×10⁶ to 5×10⁶, 5×10⁶ to 1×10⁷, 1×10⁷ to 5×10⁷, 5×10⁷ to1×10⁸, 1×10⁸ to 5×10⁸, 5×10⁸ to 1×10⁹, 1×10⁹ to 5×10⁹, 5×10⁹ to 1×10¹⁰,1×10¹⁰ to 5×10¹⁰, 5×10¹⁰ to 1×10¹¹, 5×10¹¹ to 1×10¹², 1×10¹² to 5×10¹²,and 5×10¹² to 1×10¹³.

In some embodiments, the concentration of the TILs provided in thepharmaceutical compositions of the invention is less than, for example,100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%,14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%,0.3%, 0.2%, 0.1%, 0.09% 0.08%, 0.07% 0.06% 0.05%, 0.04%, 0.03%, 0.02%,0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%,0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%,0.0002% or 0.0001% w/w, w/v or v/v of the pharmaceutical composition.

In some embodiments, the concentration of the TILs provided in thepharmaceutical compositions of the invention is greater than 90%, 80%,70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25% 19%, 18.75%,18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25% 17%, 16.75%, 16.50%, 16.25%16%, 15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%, 14.25% 14%, 13.75%,13.50%, 13.25% 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25%11%, 10.75%, 10.50%, 10.25% 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%, 8.50%,8.25% 8%, 7.75%, 7.50%, 7.25% 7%, 6.75%, 6.50%, 6.25% 6%, 5.75%, 5.50%,5.25% 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%,2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%,0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%,0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%,0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001%w/w, w/v, or v/v of the pharmaceutical composition.

In some embodiments, the concentration of the TILs provided in thepharmaceutical compositions of the invention is in the range from about0.0001% to about 50%, about 0.001% to about 40%, about 0.01% to about30%, about 0.02% to about 29%, about 0.03% to about 28%, about 0.04% toabout 27%, about 0.05% to about 26%, about 0.06% to about 25%, about0.07% to about 24%, about 0.08% to about 23%, about 0.09% to about 22%,about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% toabout 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9%to about 12% or about 1% to about 10% w/w, w/v or v/v of thepharmaceutical composition.

In some embodiments, the concentration of the TILs provided in thepharmaceutical compositions of the invention is in the range from about0.0010% to about 10%, about 0.010% to about 5%, about 0.02% to about4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% toabout 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about0.08% to about 1.5%, about 0.09% to about 1%, about 0.1% to about 0.9%w/w, w/v or v/v of the pharmaceutical composition.

In some embodiments, the amount of the TILs provided in thepharmaceutical compositions of the invention is equal to or less than 10g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g,4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g,0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g,0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008 g, 0.007g, 0.006 g, 0.005 g, 0.004 g, 0.003 g, 0.002 g, 0.001 g, 0.0009 g,0.0008 g, 0.0007 g, 0.0006 g, 0.0005 g, 0.0004 g, 0.0003 g, 0.0002 g, or0.0001 g.

In some embodiments, the amount of the TILs provided in thepharmaceutical compositions of the invention is more than 0.0001 g,0.0002 g, 0.0003 g, 0.0004 g, 0.0005 g, 0.0006 g, 0.0007 g, 0.0008 g,0.0009 g, 0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035 g, 0.004g, 0.0045 g, 0.005 g, 0.0055 g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g,0.008 g, 0.0085 g, 0.009 g, 0.0095 g, 0.01 g, 0.015 g, 0.02 g, 0.025 g,0.03 g, 0.035 g, 0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g, 0.065 g, 0.07g, 0.075 g, 0.08 g, 0.085 g, 0.09 g, 0.095 g, 0.1 g, 0.15 g, 0.2 g, 0.25g, 0.3 g, 0.35 g, 0.4 g, 0.45 g, 0.5 g, 0.55 g, 0.6 g, 0.65 g, 0.7 g,0.75 g, 0.8 g, 0.85 g, 0.9 g, 0.95 g, 1 g, 1.5 g, 2 g, 2.5, 3 g, 3.5, 4g, 4.5 g, 5 g, 5.5 g, 6 g, 6.5 g, 7 g, 7.5 g, 8 g, 8.5 g, 9 g, 9.5 g, or10 g.

The TILs provided in the pharmaceutical compositions of the inventionare effective over a wide dosage range. The exact dosage will dependupon the route of administration, the form in which the compound isadministered, the gender and age of the subject to be treated, the bodyweight of the subject to be treated, and the preference and experienceof the attending physician. The clinically-established dosages of theTILs may also be used if appropriate. The amounts of the pharmaceuticalcompositions administered using the methods herein, such as the dosagesof TILs, will be dependent on the human or mammal being treated, theseverity of the disorder or condition, the rate of administration, thedisposition of the active pharmaceutical ingredients and the discretionof the prescribing physician.

In some embodiments, TILs may be administered in a single dose. Suchadministration may be by injection, e.g., intravenous injection. In someembodiments, TILs may be administered in multiple doses. Dosing may beonce, twice, three times, four times, five times, six times, or morethan six times per year. Dosing may be once a month, once every twoweeks, once a week, or once every other day. Administration of TILs maycontinue as long as necessary.

In some embodiments, an effective dosage of TILs is about 1×10⁶, 2×10⁶,3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, 3×10⁷,4×10⁷, 5×10⁷, 6×10⁷, 7×10⁷, 8×10⁷, 9×10⁷, 1×10⁸, 2×10⁸, 3×10⁸, 4×10⁸,5×10⁸, 6×10⁸, 7×10⁸, 8×10⁸, 9×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹,6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 3×10¹⁰, 4×10¹⁰, 5×10¹⁰,6×10¹⁰, 7×10¹⁰, 8×10¹⁰, 9×10¹⁰, 1×10¹¹, 2×10¹¹, 3×10¹¹, 4×10¹¹, 5×10¹¹,6×10¹¹, 7×10¹¹, 8×10¹¹, 9×10¹¹, 1×10¹², 2×10¹², 3×10¹², 4×10¹², 5×10¹²,6×10¹², 7×10¹², 8×10¹², 9×10¹², 1×10¹³, 2×10¹³, 3×10¹³, 4×10¹³, 5×10¹³,6×10¹³, 7×10¹³, 8×10¹³, and 9×10¹³. In some embodiments, an effectivedosage of TILs is in the range of 1×10⁶ to 5×10⁶, 5×10⁶ to 1×10⁷, 1×10⁷to 5×10⁷, 5×10⁷ to 1×10⁸, 1×10⁸ to 5×10⁸, 5×10⁸ to 1×10⁹, 1×10⁹ to5×10⁹, 5×10⁹ to 1×10¹⁰, 1×10¹⁰ to 5×10¹⁰, 5×10¹⁰ to 1×10¹¹, 5×10¹¹ to1×10¹¹, 1×10¹² to 5×10¹², and 5×10¹² to 1×10¹³.

In some embodiments, an effective dosage of TILs is in the range ofabout 0.01 mg/kg to about 4.3 mg/kg, about 0.15 mg/kg to about 3.6mg/kg, about 0.3 mg/kg to about 3.2 mg/kg, about 0.35 mg/kg to about2.85 mg/kg, about 0.15 mg/kg to about 2.85 mg/kg, about 0.3 mg to about2.15 mg/kg, about 0.45 mg/kg to about 1.7 mg/kg, about 0.15 mg/kg toabout 1.3 mg/kg, about 0.3 mg/kg to about 1.15 mg/kg, about 0.45 mg/kgto about 1 mg/kg, about 0.55 mg/kg to about 0.85 mg/kg, about 0.65 mg/kgto about 0.8 mg/kg, about 0.7 mg/kg to about 0.75 mg/kg, about 0.7 mg/kgto about 2.15 mg/kg, about 0.85 mg/kg to about 2 mg/kg, about 1 mg/kg toabout 1.85 mg/kg, about 1.15 mg/kg to about 1.7 mg/kg, about 1.3 mg/kgmg to about 1.6 mg/kg, about 1.35 mg/kg to about 1.5 mg/kg, about 2.15mg/kg to about 3.6 mg/kg, about 2.3 mg/kg to about 3.4 mg/kg, about 2.4mg/kg to about 3.3 mg/kg, about 2.6 mg/kg to about 3.15 mg/kg, about 2.7mg/kg to about 3 mg/kg, about 2.8 mg/kg to about 3 mg/kg, or about 2.85mg/kg to about 2.95 mg/kg.

In some embodiments, an effective dosage of TILs is in the range ofabout 1 mg to about 500 mg, about 10 mg to about 300 mg, about 20 mg toabout 250 mg, about 25 mg to about 200 mg, about 1 mg to about 50 mg,about 5 mg to about 45 mg, about 10 mg to about 40 mg, about 15 mg toabout 35 mg, about 20 mg to about 30 mg, about 23 mg to about 28 mg,about 50 mg to about 150 mg, about 60 mg to about 140 mg, about 70 mg toabout 130 mg, about 80 mg to about 120 mg, about 90 mg to about 110 mg,or about 95 mg to about 105 mg, about 98 mg to about 102 mg, about 150mg to about 250 mg, about 160 mg to about 240 mg, about 170 mg to about230 mg, about 180 mg to about 220 mg, about 190 mg to about 210 mg,about 195 mg to about 205 mg, or about 198 to about 207 mg.

An effective amount of the TILs may be administered in either single ormultiple doses by any of the accepted modes of administration of agentshaving similar utilities, including intranasal and transdermal routes,by intra-arterial injection, intravenously, intraperitoneally,parenterally, intramuscularly, subcutaneously, topically, bytransplantation, or by inhalation.

H. Cryopreservation of TILs

As discussed above, and exemplified in Steps A through E as provided inFIG. 9, cryopreservation can occur at numerous points throughout the TILexpansion process. In some embodiments, the expanded population of TILsafter the second expansion (as provided for example, according to Step Dof FIG. 9) can be cryopreserved. Cryopreservation can be generallyaccomplished by placing the TIL population into a freezing solution,e.g., 85% complement inactivated AB serum and 15% dimethyl sulfoxide(DMSO). The cells in solution are placed into cryogenic vials and storedfor 24 hours at −80° C., with optional transfer to gaseous nitrogenfreezers for cryopreservation. See Sadeghi, et al., Acta Oncologica2013, 52, 978-986. In some embodiments, the TILs are cryopreserved in 5%DMSO. In some embodiments, the TILs are cryopreserved in cell culturemedia plus 5% DMSO. In some embodiments, the TILs are cryopreservedaccording to the methods provided in Examples 8 and 9.

When appropriate, the cells are removed from the freezer and thawed in a37° C. water bath until approximately 4/5 of the solution is thawed. Thecells are generally resuspended in complete media and optionally washedone or more times. In some embodiments, the thawed TILs can be countedand assessed for viability as is known in the art.

I. Closed Systems for TIL Manufacturing

The present invention provides for the use of closed systems during theTIL culturing process. Such closed systems allow for preventing and/orreducing microbial contamination, allow for the use of fewer flasks, andallow for cost reductions. In some embodiments, the closed system usestwo containers.

Such closed systems are well-known in the art and can be found, forexample, at http://www.fda.gov/cber/guidelines.htm andhttps://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatorylnformation/Guidances/Blood/ucm076779.htm.

As provided on the FDA website, closed systems with sterile methods areknown and well described. See,https://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatorylnformation/Guidances/Blood/ucm076779.htm, as referenced above and provided inpertinent part below.

Sterile connecting devices (STCDs) produce sterile welds between twopieces of compatible tubing. This procedure permits sterile connectionof a variety of containers and tube diameters. This guidance describesrecommended practices and procedures for use of these devices. Thisguidance does not address the data or information that a manufacturer ofa sterile connecting device must submit to FDA in order to obtainapproval or clearance for marketing. It is also important to note thatthe use of an approved or cleared sterile connecting device for purposesnot authorized in the labeling may cause the device to be consideredadulterated and misbranded under the Federal Food, Drug and CosmeticAct.

In some embodiments, the closed system uses one container from the timethe tumor fragments are obtained until the TILs are ready foradministration to the patient or cryopreserving. In some embodimentswhen two containers are used, the first container is a closedG-container and the population of TILs is centrifuged and transferred toan infusion bag without opening the first closed G-container. In someembodiments, when two containers are used, the infusion bag is aHypoThermosol-containing infusion bag. A closed system or closed TILcell culture system is characterized in that once the tumor sampleand/or tumor fragments have been added, the system is tightly sealedfrom the outside to form a closed environment free from the invasion ofbacteria, fungi, and/or any other microbial contamination.

In some embodiments, the reduction in microbial contamination is betweenabout 5% and about 100%. In some embodiments, the reduction in microbialcontamination is between about 5% and about 95%. In some embodiments,the reduction in microbial contamination is between about 5% and about90%. In some embodiments, the reduction in microbial contamination isbetween about 10% and about 90%. In some embodiments, the reduction inmicrobial contamination is between about 15% and about 85%. In someembodiments, the reduction in microbial contamination is about 5%, about10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%,about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%,about 99%, or about 100%.

The closed system allows for TIL growth in the absence and/or with asignificant reduction in microbial contamination.

Moreover, pH, carbon dioxide partial pressure and oxygen partialpressure of the TIL cell culture environment each vary as the cells arecultured. Consequently, even though a medium appropriate for cellculture is circulated, the closed environment still needs to beconstantly maintained as an optimal environment for TIL proliferation.To this end, it is desirable that the physical factors of pH, carbondioxide partial pressure and oxygen partial pressure within the cultureliquid of the closed environment be monitored by means of a sensor, thesignal whereof is used to control a gas exchanger installed at the inletof the culture environment, and the that gas partial pressure of theclosed environment be adjusted in real time according to changes in theculture liquid so as to optimize the cell culture environment. In someembodiments, the present invention provides a closed cell culture systemwhich incorporates at the inlet to the closed environment a gasexchanger equipped with a monitoring device which measures the pH,carbon dioxide partial pressure and oxygen partial pressure of theclosed environment, and optimizes the cell culture environment byautomatically adjusting gas concentrations based on signals from themonitoring device.

In some embodiments, the pressure within the closed environment iscontinuously or intermittently controlled. That is, the pressure in theclosed environment can be varied by means of a pressure maintenancedevice for example, thus ensuring that the space is suitable for growthof TILs in a positive pressure state, or promoting exudation of fluid ina negative pressure state and thus promoting cell proliferation. Byapplying negative pressure intermittently, moreover, it is possible touniformly and efficiently replace the circulating liquid in the closedenvironment by means of a temporary shrinkage in the volume of theclosed environment.

In some embodiments, optimal culture components for proliferation of theTILs can be substituted or added, and including factors such as IL-2and/or OKT3, as well as combination, can be added.

C. Cell Cultures

In an embodiment, a method for expanding TILs, including those discussabove as well as exemplified in FIG. 9, may include using about 5,000 mLto about 25,000 mL of cell medium, about 5,000 mL to about 10,000 mL ofcell medium, or about 5,800 mL to about 8,700 mL of cell medium. In someembodiments, the media is a serum free medium. In some embodiments, themedia in the first expansion is serum free. In some embodiments, themedia in the second expansion is serum free. In some embodiments, themedia in the first expansion and the second are both serum free. In anembodiment, expanding the number of TILs uses no more than one type ofcell culture medium. Any suitable cell culture medium may be used, e.g.,AIM-V cell medium (L-glutamine, 50 μM streptomycin sulfate, and 10 μMgentamicin sulfate) cell culture medium (Invitrogen, Carlsbad Calif.).In this regard, the inventive methods advantageously reduce the amountof medium and the number of types of medium required to expand thenumber of TIL. In an embodiment, expanding the number of TIL maycomprise feeding the cells no more frequently than every third or fourthday. Expanding the number of cells in a gas permeable containersimplifies the procedures necessary to expand the number of cells byreducing the feeding frequency necessary to expand the cells.

In an embodiment, the cell medium in the first and/or second gaspermeable container is unfiltered. The use of unfiltered cell medium maysimplify the procedures necessary to expand the number of cells. In anembodiment, the cell medium in the first and/or second gas permeablecontainer lacks beta-mercaptoethanol (BME).

In an embodiment, the duration of the method comprising obtaining atumor tissue sample from the mammal; culturing the tumor tissue samplein a first gas permeable container containing cell medium therein;obtaining TILs from the tumor tissue sample; expanding the number ofTILs in a second gas permeable container containing cell medium for aduration of about 7 to 14 days, e.g., about 11 days. In some embodimentspre-REP is about 7 to 14 days, e.g., about 11 days. In some embodiments,REP is about 7 to 14 days, e.g., about 11 days.

In an embodiment, TILs are expanded in gas-permeable containers.Gas-permeable containers have been used to expand TILs using PBMCs usingmethods, compositions, and devices known in the art, including thosedescribed in U.S. Patent Application Publication No. 2005/0106717 A1,the disclosures of which are incorporated herein by reference. In anembodiment, TILs are expanded in gas-permeable bags. In an embodiment,TILs are expanded using a cell expansion system that expands TILs in gaspermeable bags, such as the Xuri Cell Expansion System W25 (GEHealthcare). In an embodiment, TILs are expanded using a cell expansionsystem that expands TILs in gas permeable bags, such as the WAVEBioreactor System, also known as the Xuri Cell Expansion System W5 (GEHealthcare). In an embodiment, the cell expansion system includes a gaspermeable cell bag with a volume selected from the group consisting ofabout 100 mL, about 200 mL, about 300 mL, about 400 mL, about 500 mL,about 600 mL, about 700 mL, about 800 mL, about 900 mL, about 1 L, about2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L,about 9 L, and about 10 L.

In an embodiment, TILs can be expanded in G-Rex flasks (commerciallyavailable from Wilson Wolf Manufacturing). Such embodiments allow forcell populations to expand from about 5×10⁵ cells/cm² to between 10×10⁶and 30×10⁶ cells/cm². In an embodiment this is without feeding. In anembodiment, this is without feeding so long as medium resides at aheight of about 10 cm in the G-Rex flask. In an embodiment this iswithout feeding but with the addition of one or more cytokines. In anembodiment, the cytokine can be added as a bolus without any need to mixthe cytokine with the medium. Such containers, devices, and methods areknown in the art and have been used to expand TILs, and include thosedescribed in U.S. Patent Application Publication No. US 2014/0377739A1,International Publication No. WO 2014/210036 A1, U.S. Patent ApplicationPublication No. us 2013/0115617 A1, International Publication No. WO2013/188427 A1, U.S. Patent Application Publication No. US 2011/0136228A1, U.S. Pat. No. 8,809,050 B2, International publication No. WO2011/072088 A2, U.S. Patent Application Publication No. US 2016/0208216A1, U.S. Patent Application Publication No. US 2012/0244133 A1,International Publication No. WO 2012/129201 A1, U.S. Patent ApplicationPublication No. US 2013/0102075 A1, U.S. Pat. No. 8,956,860 B2,International Publication No. WO 2013/173835 A1, U.S. Patent ApplicationPublication No. US 2015/0175966 A1, the disclosures of which areincorporated herein by reference. Such processes are also described inJin et al., J. Immunotherapy, 2012, 35:283-292.

D. Optional Cryopreservation of TILs

Either the bulk TIL population or the expanded population of TILs can beoptionally cryopreserved. In some embodiments, cryopreservation occurson the therapeutic TIL population. In some embodiments, cryopreservationoccurs on the TILs harvested after the second expansion. In someembodiments, cryopreservation occurs on the TILs in exemplary Step F ofFIG. 9. In some embodiments, the TILs are cryopreserved in the infusionbag. In some embodiments, the TILs are cryopreserved prior to placementin an infusion bag. In some embodiments, the TILs are cryopreserved andnot placed in an infusion bag. In some embodiments, cryopreservation isperformed using a cryopreservation medium. In some embodiments, thecryopreservation media contains dimethylsulfoxide (DMSO). This isgenerally accomplished by putting the TIL population into a freezingsolution, e.g. 85% complement inactivated AB serum and 15% dimethylsulfoxide (DMSO). The cells in solution are placed into cryogenic vialsand stored for 24 hours at −80° C., with optional transfer to gaseousnitrogen freezers for cryopreservation. See, Sadeghi, et al., ActaOncologica 2013, 52, 978-986.

When appropriate, the cells are removed from the freezer and thawed in a37° C. water bath until approximately 4/5 of the solution is thawed. Thecells are generally resuspended in complete media and optionally washedone or more times. In some embodiments, the thawed TILs can be countedand assessed for viability as is known in the art.

In a preferred embodiment, a population of TILs is cryopreserved usingCS10 cryopreservation media (CryoStor 10, BioLife Solutions). In apreferred embodiment, a population of TILs is cryopreserved using acryopreservation media containing dimethylsulfoxide (DMSO). In apreferred embodiment, a population of TILs is cryopreserved using a 1:1(vol:vol) ratio of CS10 and cell culture media. In a preferredembodiment, a population of TILs is cryopreserved using about a 1:1(vol:vol) ratio of CS10 and cell culture media, further comprisingadditional IL-2.

According to particular embodiments, a cryopreservation composition(also referred to herein as a “dimethylsulfoxide-based cryopreservationmedium”) comprises a population of TILs prepared in accordance with thepresent invention (e.g., in an amount of 1×10⁶ to 9×10¹³), acryoprotectant medium comprising DMSO that is suitable for preservingcells in low-temperature environments such as −70° C. to −196° C. (e.g.,CryoStor® CS10) and an electrolyte solution (e.g., an isotonic solutionsuch as PlasmaLyte® A). In a preferred embodiment, the cryoprotectantmedium and electrolyte solution are present in a ratio of between about1.2:1 and about 1:1.2, or between about 1.1:1 and about 1:1.1, orpreferably about 1:1. According to one embodiment, the electrolytesolution comprises one or more of sodium, potassium, magnesium, acetate,chloride and gluconate, or a combination thereof, for example, theelectrolyte solution may comprise sodium chloride, sodium gluconate,sodium acetate trihydrate, potassium chloride and magnesium chloride.According to an embodiment, the electrolyte solution has a pH betweenabout 7 and about 8, preferably between about 7.2 and about 7.6, orabout 7.4. Preferably, the cryopreservation composition furthercomprises one or more stabilizers (e.g., human serum albumin) and/or oneor more lymphocyte growth factors (e.g., IL-2). For example, each of thecryoprotectant medium and the electrolyte solution may be present in thecryopreservation composition in an amount of about 20 mL to about 100mL, or about 30 mL to about 70 mL, or about 40 mL to about 60 mL, orabout 50 mL; human serum albumin may be present in an amount of about0.01 g to about 2.0 g, or about 0.1 g to about 1.0 g, or about 0.5 g;and IL-2 may be present in an amount of about 0.001 mg to about 0.005mg, or about 0.0015 mg to about 0.0025 mg, or about 0.0018 mg. Thecryopreservation medium may optionally comprise one or more additionaladditives or excipients, such as pH adjusters, preservatives, etc.

According to one embodiment, a cryopreservation composition containing apopulation of TILs is composed of the following:

Nominal Composition (% v/V) of Drug Product (100 mL basis) IngredientNominal Quantity per 100 mL fill Function Tumor Infiltrating Lymphocytes83 × 10⁹ to 3.0 × 10¹⁰ Active Ingredient CryoStor ® CS10^(a)    50 mLCryopreservation medium PlasmaLyte A    50 mL Isotonic agent Human SerumAlbumin (HSA)   0.5 g Stabilizer Interleukin-2 0.0018 mg (30,000 IU)Lymphocyte growth factor Total   100 mL ^(a)Cryostor CS10 contains 10%dimethylsulfoxide (DMSO)

As discussed above in Steps A through E, cryopreservation can occur atnumerous points throughout the TIL expansion process. In someembodiments, the bulk TIL population after the first expansion accordingto Step B or the expanded population of TILs after the one or moresecond expansions according to Step D can be cryopreserved.Cryopreservation can be generally accomplished by placing the TILpopulation into a freezing solution, e.g., 85% complement inactivated ABserum and 15% dimethyl sulfoxide (DMSO). The cells in solution areplaced into cryogenic vials and stored for 24 hours at −80° C., withoptional transfer to gaseous nitrogen freezers for cryopreservation. SeeSadeghi, et al., Acta Oncologica 2013, 52, 978-986.

When appropriate, the cells are removed from the freezer and thawed in a37° C. water bath until approximately 4/5 of the solution is thawed. Thecells are generally resuspended in complete media and optionally washedone or more times. In some embodiments, the thawed TILs can be countedand assessed for viability as is known in the art.

In some cases, the Step B TIL population can be cryopreservedimmediately, using the protocols discussed below. Alternatively, thebulk TIL population can be subjected to Step C and Step D and thencryopreserved after Step D. Similarly, in the case where geneticallymodified TILs will be used in therapy, the Step B or Step D TILpopulations can be subjected to genetic modifications for suitabletreatments.

V. Methods of Treating Cancers and Other Diseases

The compositions and methods described herein can be used in a methodfor treating diseases. In an embodiment, they are for use in treatinghyperproliferative disorders. They may also be used in treating otherdisorders as described herein and in the following paragraphs.

In some embodiments, the hyperproliferative disorder is cancer. In someembodiments, the hyperproliferative disorder is a solid tumor cancer. Insome embodiments, the solid tumor cancer is selected from the groupconsisting of melanoma, ovarian cancer, cervical cancer, non-small-celllung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancercaused by human papilloma virus, head and neck cancer (including headand neck squamous cell carcinoma (HNSCC)), renal cancer, and renal cellcarcinoma. In some embodiments, the hyperproliferative disorder is ahematological malignancy. In some embodiments, the solid tumor cancer isselected from the group consisting of chronic lymphocytic leukemia,acute lymphoblastic leukemia, diffuse large B cell lymphoma,non-Hodgkin's lymphoma, Hodgkin's lymphoma, follicular lymphoma, andmantle cell lymphoma.

In an embodiment, the invention includes a method of treating a cancerwith a population of TILs, wherein a patient is pre-treated withnon-myeloablative chemotherapy prior to an infusion of TILs according tothe present disclosure. In an embodiment, the non-myeloablativechemotherapy is cyclophosphamide 60 mg/kg/d for 2 days (days 27 and 26prior to TIL infusion) and fludarabine 25 mg/m²/d for 5 days (days 27 to23 prior to TIL infusion). In an embodiment, after non-myeloablativechemotherapy and TIL infusion (at day 0) according to the presentdisclosure, the patient receives an intravenous infusion of IL-2intravenously at 720,000 IU/kg every 8 hours to physiologic tolerance.

Efficacy of the TILs described herein in treating, preventing and/ormanaging the indicated diseases or disorders can be tested using variousmodels known in the art, which provide guidance for treatment of humandisease. For example, models for determining efficacy of treatments forovarian cancer are described, e.g., in Mullany, et al., Endocrinology2012, 153, 1585-92; and Fong, et al., J Ovarian Res. 2009, 2, 12. Modelsfor determining efficacy of treatments for pancreatic cancer aredescribed in Herreros-Villanueva, et al., World J Gastroenterol. 2012,18, 1286-1294. Models for determining efficacy of treatments for breastcancer are described, e.g., in Fantozzi, Breast Cancer Res. 2006, 8,212. Models for determining efficacy of treatments for melanoma aredescribed, e.g., in Damsky, et al., Pigment Cell & Melanoma Res. 2010,23, 853-859. Models for determining efficacy of treatments for lungcancer are described, e.g., in Meuwissen, et al., Genes & Development,2005, 19, 643-664. Models for determining efficacy of treatments forlung cancer are described, e.g., in Kim, Clin. Exp. Otorhinolaryngol.2009, 2, 55-60; and Sano, Head Neck Oncol. 2009, 1, 32.

In some embodiments, IFN-gamma (IFN-γ) is indicative of treatmentefficacy for hyperproliferative disorder treatment. In some embodiments,IFN-γ in the blood of subjects treated with TILs is indicative of activeTILs. In some embodiments, a potency assay for IFN-γ production isemployed. IFN-γ production is another measure of cytotoxic potential.IFN-γ production can be measured by determining the levels of thecytokine IFN-γ in the blood of a subject treated with TILs prepared bythe methods of the present invention, including those as described forexample in FIG. 20 or FIG. 21. In some embodiments, the TILs obtained bythe present method provide for increased IFN-γ in the blood of subjectstreated with the TILs of the present method as compared to subjectstreated with TILs prepared using methods referred to as process 1C, asexemplified in FIG. 13. In some embodiments, an increase in IFN-γ isindicative of treatment efficacy in a patient treated with the TILsproduced by the methods of the present invention. In some embodiments,IFN-γ is increased one-fold, two-fold, three-fold, four-fold, orfive-fold or more as compared to an untreated patient and/or as comparedto a patient treated with TILs prepared using other methods than thoseprovide herein including for example, methods other than those embodiedin FIG. 20 or FIG. 21. In some embodiments, IFN-γ secretion is increasedone-fold as compared to an untreated patient and/or as compared to apatient treated with TILs prepared using other methods than thoseprovide herein including for example, methods other than those embodiedin FIG. 20 or FIG. 21. In some embodiments, IFN-γ secretion is increasedtwo-fold as compared to an untreated patient and/or as compared to apatient treated with TILs prepared using other methods than thoseprovide herein including for example, methods other than those embodiedin FIG. 20 or FIG. 21. In some embodiments, IFN-γ secretion is increasedthree-fold as compared to an untreated patient and/or as compared to apatient treated with TILs prepared using other methods than thoseprovide herein including for example, methods other than those embodiedin FIG. 20 or FIG. 21. In some embodiments, IFN-γ secretion is increasedfour-fold as compared to an untreated patient and/or as compared to apatient treated with TILs prepared using other methods than thoseprovide herein including for example, methods other than those embodiedin FIG. 20 or FIG. 21. In some embodiments, IFN-γ secretion is increasedfive-fold as compared to an untreated patient and/or as compared to apatient treated with TILs prepared using other methods than thoseprovide herein including for example, methods other than those embodiedin FIG. 20 or FIG. 21. In some embodiments, IFN-γ is measured using aQuantikine ELISA kit. In some embodiments, IFN-γ is measured using aQuantikine ELISA kit. In some embodiments, IFN-γ is measured in TILs exvivo from a patient treated with the TILs produced by the methods of thepresent invention. In some embodiments, IFN-γ is measured in blood in apatient treated with the TILs produced by the methods of the presentinvention. In some embodiments, IFN-γ is measured in serum in a patienttreated with the TILs produced by the methods of the present invention.

In some embodiments, the TILs prepared by the methods of the presentinvention, including those as described for example in FIG. 20 or FIG.21, exhibit increased polyclonality as compared to TILs produced byother methods, including those not exemplified in FIG. 20 or FIG. 21,such as for example, methods referred to as process 1C methods. In someembodiments, significantly improved polyclonality and/or increasedpolyclonality is indicative of treatment efficacy and/or increasedclinical efficacy for cancer treatment. In some embodiments,polyclonality refers to the T-cell repertoire diversity. In someembodiments, an increase in polyclonality can be indicative of treatmentefficacy with regard to administration of the TILs produced by themethods of the present invention. In some embodiments, polyclonality isincreased one-fold, two-fold, ten-fold, 100-fold, 500-fold, or 1000-foldas compared to TILs prepared using methods than those provide hereinincluding for example, methods other than those embodied in FIG. 20 orFIG. 21. In some embodiments, polyclonality is increased one-fold ascompared to an untreated patient and/or as compared to a patient treatedwith TILs prepared using other methods than those provide hereinincluding for example, methods other than those embodied in FIG. 9. Insome embodiments, polyclonality is increased two-fold as compared to anuntreated patient and/or as compared to a patient treated with TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 9. In someembodiments, polyclonality is increased ten-fold as compared to anuntreated patient and/or as compared to a patient treated with TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 9. In someembodiments, polyclonality is increased 100-fold as compared to anuntreated patient and/or as compared to a patient treated with TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 9. In someembodiments, polyclonality is increased 500-fold as compared to anuntreated patient and/or as compared to a patient treated with TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 9. In someembodiments, polyclonality is increased 1000-fold as compared to anuntreated patient and/or as compared to a patient treated with TILsprepared using other methods than those provide herein including forexample, methods other than those embodied in FIG. 9.

1. Methods of Co-Administration

In some embodiments, the TILs produced as described herein, includingfor example TILs derived from a method described in FIG. 20 or FIG. 21,can be administered in combination with one or more immune checkpointregulators, such as the antibodies described below. For example,antibodies that target PD-1 and which can be co-administered with theTILs of the present invention include, e.g., but are not limited tonivolumab (BMS-936558, Bristol-Myers Squibb; Opdivo®), pembrolizumab(lambrolizumab, MK03475 or MK-3475, Merck; Keytruda®), humanizedanti-PD-1 antibody JS001 (ShangHai JunShi), monoclonal anti-PD-1antibody TSR-042 (Tesaro, Inc.), Pidilizumab (anti-PD-1 mAb CT-011,Medivation), anti-PD-1 monoclonal Antibody BGB-A317 (BeiGene), and/oranti-PD-1 antibody SHR-1210 (ShangHai HengRui), human monoclonalantibody REGN2810 (Regeneron), human monoclonal antibody MDX-1106(Bristol-Myers Squibb), and/or humanized anti-PD-1 IgG4 antibody PDR001(Novartis). In some embodiments, the PD-1 antibody is from clone:RMP1-14 (rat IgG)—BioXcell cat# BP0146. Other suitable antibodiessuitable for use in co-administration methods with TILs producedaccording to Steps A through F as described herein are anti-PD-1antibodies disclosed in U.S. Pat. No. 8,008,449, herein incorporated byreference. In some embodiments, the antibody or antigen-binding portionthereof binds specifically to PD-L1 and inhibits its interaction withPD-1, thereby increasing immune activity. Any antibodies known in theart which bind to PD-L1 and disrupt the interaction between the PD-1 andPD-L1, and stimulates an anti-tumor immune response, are suitable foruse in co-administration methods with TILs produced according to Steps Athrough F as described herein. For example, antibodies that target PD-L1and are in clinical trials, include BMS-936559 (Bristol-Myers Squibb)and MPDL3280A (Genentech). Other suitable antibodies that target PD-L1are disclosed in U.S. Pat. No. 7,943,743, herein incorporated byreference. It will be understood by one of ordinary skill that anyantibody which binds to PD-1 or PD-L1, disrupts the PD-1/PD-L1interaction, and stimulates an anti-tumor immune response, are suitablefor use in co-administration methods with TILs produced according toSteps A through F as described herein. In some embodiments, the subjectadministered the combination of TILs produced according to Steps Athrough F is co administered with a and anti-PD-1 antibody when thepatient has a cancer type that is refractory to administration of theanti-PD-1 antibody alone. In some embodiments, the patient isadministered TILs in combination with and anti-PD-1 when the patient hasrefractory melanoma. In some embodiments, the patient is administeredTILs in combination with and anti-PD-1 when the patient hasnon-small-cell lung carcinoma (NSCLC).

2. Optional Lymphodepletion Preconditioning of Patients

In an embodiment, the invention includes a method of treating a cancerwith a population of TILs, wherein a patient is pre-treated withnon-myeloablative chemotherapy prior to an infusion of TILs according tothe present disclosure. In an embodiment, the invention includes apopulation of TILs for use in the treatment of cancer in a patient whichhas been pre-treated with non-myeloablative chemotherapy. In anembodiment, the population of TILs is for administration by infusion. Inan embodiment, the non-myeloablative chemotherapy is cyclophosphamide 60mg/kg/d for 2 days (days 27 and 26 prior to TIL infusion) andfludarabine 25 mg/m²/d for 5 days (days 27 to 23 prior to TIL infusion).In an embodiment, after non-myeloablative chemotherapy and TIL infusion(at day 0) according to the present disclosure, the patient receives anintravenous infusion of IL-2 (aldesleukin, commercially available asPROLEUKIN) intravenously at 720,000 IU/kg every 8 hours to physiologictolerance. In certain embodiments, the population of TILs is for use intreating cancer in combination with IL-2, wherein the IL-2 isadministered after the population of TILs.

Experimental findings indicate that lymphodepletion prior to adoptivetransfer of tumor-specific T lymphocytes plays a key role in enhancingtreatment efficacy by eliminating regulatory T cells and competingelements of the immune system (‘cytokine sinks’). Accordingly, someembodiments of the invention utilize a lymphodepletion step (sometimesalso referred to as “immunosuppressive conditioning”) on the patientprior to the introduction of the TILs of the invention.

In general, lymphodepletion is achieved using administration offludarabine or cyclophosphamide (the active form being referred to asmafosfamide) and combinations thereof. Such methods are described inGassner, et al., Cancer Immunol. Immunother. 2011, 60, 75-85, Muranski,et al., Nat. C/in. Pract. Oncol., 2006, 3, 668-681, Dudley, et al., J.Clin. Oncol. 2008, 26, 5233-5239, and Dudley, et al., J. Clin. Oncol.2005, 23, 2346-2357, all of which are incorporated by reference hereinin their entireties.

In some embodiments, the fludarabine is administered at a concentrationof 0.5 μg/mL to 10 μg/mL fludarabine. In some embodiments, thefludarabine is administered at a concentration of 1 μg/mL fludarabine.In some embodiments, the fludarabine treatment is administered for 1day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or more. In someembodiments, the fludarabine is administered at a dosage of 10mg/kg/day, 15 mg/kg/day, 20 mg/kg/day, 25 mg/kg/day, 30 mg/kg/day, 35mg/kg/day, 40 mg/kg/day, or 45 mg/kg/day. In some embodiments, thefludarabine treatment is administered for 2-7 days at 35 mg/kg/day. Insome embodiments, the fludarabine treatment is administered for 4-5 daysat 35 mg/kg/day. In some embodiments, the fludarabine treatment isadministered for 4-5 days at 25 mg/kg/day.

In some embodiments, the mafosfamide, the active form ofcyclophosphamide, is obtained at a concentration of 0.5 μg/mL-10 μg/mLby administration of cyclophosphamide. In some embodiments, mafosfamide,the active form of cyclophosphamide, is obtained at a concentration of 1μg/mL by administration of cyclophosphamide. In some embodiments, thecyclophosphamide treatment is administered for 1 day, 2 days, 3 days, 4days, 5 days, 6 days, or 7 days or more. In some embodiments, thecyclophosphamide is administered at a dosage of 100 mg/m²/day, 150mg/m²/day, 175 mg/m²/day, 200 mg/m²/day, 225 mg/m²/day, 250 mg/m²/day,275 mg/m²/day, or 300 mg/m²/day. In some embodiments, thecyclophosphamide is administered intravenously (i.e., i.v.) In someembodiments, the cyclophosphamide treatment is administered for 2-7 daysat 35 mg/kg/day. In some embodiments, the cyclophosphamide treatment isadministered for 4-5 days at 250 mg/m²/day i.v. In some embodiments, thecyclophosphamide treatment is administered for 4 days at 250 mg/m²/dayi.v.

In some embodiments, lymphodepletion is performed by administering thefludarabine and the cyclophosphamide together to a patient. In someembodiments, fludarabine is administered at 25 mg/m²/day i.v. andcyclophosphamide is administered at 250 mg/m²/day i.v. over 4 days.

In an embodiment, the lymphodepletion is performed by administration ofcyclophosphamide at a dose of 60 mg/m²/day for two days followed byadministration of fludarabine at a dose of 25 mg/m²/day for five days.

3. IL-2 Regimens

In an embodiment, the IL-2 regimen comprises a high-dose IL-2 regimen,wherein the high-dose IL-2 regimen comprises aldesleukin, or abiosimilar or variant thereof, administered intravenously starting onthe day after administering a therapeutically effective portion of thetherapeutic population of TILs, wherein the aldesleukin or a biosimilaror variant thereof is administered at a dose of 0.037 mg/kg or 0.044mg/kg IU/kg (patient body mass) using 15-minute bolus intravenousinfusions every eight hours until tolerance, for a maximum of 14 doses.Following 9 days of rest, this schedule may be repeated for another 14doses, for a maximum of 28 doses in total.

In an embodiment, the IL-2 regimen comprises a decrescendo IL-2 regimen.Decrescendo IL-2 regimens have been described in O'Day, et al., J Clin.Oncol. 1999, 17, 2752-61 and Eton, et al., Cancer 2000, 88, 1703-9, thedisclosures of which are incorporated herein by reference. In anembodiment, a decrescendo IL-2 regimen comprises 18×10⁶ IU/m²administered intravenously over 6 hours, followed by 18×10⁶ IU/m²administered intravenously over 12 hours, followed by 18×10⁶ IU/m²administered intravenously over 24 hrs, followed by 4.5×10⁶ IU/m²administered intravenously over 72 hours. This treatment cycle may berepeated every 28 days for a maximum of four cycles. In an embodiment, adecrescendo IL-2 regimen comprises 18,000,000 IU/m² on day 1, 9,000,000IU/m² on day 2, and 4,500,000 IU/m² on days 3 and 4.

In an embodiment, the IL-2 regimen comprises administration of pegylatedIL-2 every 1, 2, 4, 6, 7, 14 or 21 days at a dose of 0.10 mg/day to 50mg/day.

4. Adoptive Cell Transfer

Adoptive cell transfer (ACT) is a very effective form of immunotherapyand involves the transfer of immune cells with antitumor activity intocancer patients. ACT is a treatment approach that involves theidentification, in vitro, of lymphocytes with antitumor activity, the invitro expansion of these cells to large numbers and their infusion intothe cancer-bearing host. Lymphocytes used for adoptive transfer can bederived from the stroma of resected tumors (tumor infiltratinglymphocytes or TILs). TILs for ACT can be prepared as described herein.In some embodiments, the TILs are prepared, for example, according to amethod as described in FIG. 9. They can also be derived or from blood ifthey are genetically engineered to express antitumor T-cell receptors(TCRs) or chimeric antigen receptors (CARs), enriched with mixedlymphocyte tumor cell cultures (MLTCs), or cloned using autologousantigen presenting cells and tumor derived peptides. ACT in which thelymphocytes originate from the cancer-bearing host to be infused istermed autologous ACT. U.S. Publication No. 2011/0052530 relates to amethod for performing adoptive cell therapy to promote cancerregression, primarily for treatment of patients suffering frommetastatic melanoma, which is incorporated by reference in its entiretyfor these methods. In some embodiments, TILs can be administered asdescribed herein. In some embodiments, TILs can be administered in asingle dose. Such administration may be by injection, e.g., intravenousinjection. In some embodiments, TILs and/or cytotoxic lymphocytes may beadministered in multiple doses. Dosing may be once, twice, three times,four times, five times, six times, or more than six times per year.Dosing may be once a month, once every two weeks, once a week, or onceevery other day. Administration of TILs and/or cytotoxic lymphocytes maycontinue as long as necessary.

EXAMPLES

The embodiments encompassed herein are now described with reference tothe following examples. These examples are provided for the purpose ofillustration only and the disclosure encompassed herein should in no waybe construed as being limited to these examples, but rather should beconstrued to encompass any and all variations which become evident as aresult of the teachings provided herein.

Example 1. Production of a Cryopreserved TIL Therapy

This example describes the cGMP manufacture of TIL therapy in G-RexFlasks according to current Good Tissue Practices and current GoodManufacturing Practices.

Process Reference Expansion Plan

Estimated Day (post- Estimated Total seed) Activity Target CriteriaAnticipated Vessels Volume (mL) 0 Tumor Dissection ≤50 desirable tumorfragments per G- G-Rex100MCS 1 flask ≤1000 Rex100MCS 11 REP Seed 5 − 200× 10⁶ viable cells per G- G-Rex500MCS 1 flasks ≤5000 Rex500MCS 16 REPSplit 1 × 10⁹ viable cells per G-Rex500MCS ≤5 ≤25000 G-Rex500MCS flasks22 Harvest Total available cells 3-4 CS-750 bags ≤530

Flask Volumes:

Working Volume/Flask Flask Type (mL) G-Rex100MCS 1000 G-Rex500MCS 5000

Equipment

Equipment List: Day 0 CM1 Media Preparation/Tumor Wash Preparation/TumorDissection:

-   -   Magnehelic Gauge    -   Biological Safety Cabinet (BSC)    -   Incubator    -   CO₂ Analyzer    -   Micropipetter (100-1000 μL)    -   Pipet-Aid    -   Baxa Repeater Pump    -   Sebra Tube Sealer    -   2-8° C. Refrigerator    -   −80° C. Freezer    -   −20° C. Freezer    -   Timer

Equipment List: CM2 Preparation/Day 11 REP Seed

-   -   Magnehelic Gauge    -   Biological Safety Cabinet (BSC)    -   Incubator    -   Incubator    -   CO2 Analyzer    -   Dry Bath    -   Water Bath    -   CytoTherm    -   Welder    -   Gatherex    -   NC200 NucleoCounter    -   Baxa Repeater Pump    -   Sebra Tube Sealer Balance

Equipment List: CM2 Preparation/Day 11 REP Seed

-   -   Centrifuge    -   Micropipetter (100-1000 μL)    -   Pipet-Aid    -   Timer    -   2-8° C. Refrigerator    -   −80° C. Freezer    -   Controlled Rate Freezer    -   LN2 Storage Freezer (Quarantine)    -   −20° C. Freezer

Equipment List: CM4 Preparation/Day 16

-   -   Magnehelic Gauge    -   Biological Safety Cabinet (BSC)    -   Incubator    -   Incubator    -   CO2 Analyzer    -   Welder    -   Welder    -   Gatherex    -   NC200 NucleoCounter    -   Baxa Repeater Pump    -   Sebra Tube Sealer Balance    -   Micropipetter (100-1000 μL)    -   Pipet-Aid    -   2-8° C. Refrigerator    -   −80° C. Freezer

Equipment List: Day 22 Formulation, Fill, Cryopreservation

-   -   Magnehelic Gauge    -   Biological Safety Cabinet (BSC)    -   Incubator    -   Incubator    -   CO2 Analyzer    -   Welder    -   Gatherex    -   NC200 NucleoCounter    -   Baxa Repeater Pump    -   Sebra Tube Sealer Balance    -   Micropipetter (20-200 μL) Pipet-Aid

Equipment List: Day 22 Formulation, Fill, Cryopreservation

-   -   Pipet-Aid    -   2-8° C. Refrigerator    -   −80° C. Freezer    -   Controlled Rate Freezer    -   LN2 Storage Freezer    -   LN2 Storage Freezer (Quarantine)    -   LOVO Cell Processing System

7.0 Materials

Materials: Day 0 CM1 Media Preparation/Tumor Wash Preparation/Tumor

Dissection

-   -   Disposable Scalpels, Sterile    -   50 mL Serological Pipets, Sterile    -   1 mL Serological Plastic Pipet, Sterile    -   10 mL Serological Pipet, Sterile    -   Centrifuge Tube, 50 mL, 28×114 mm, Conical Base, Screw Cap, PP,        Sterile    -   25 mL Serological Pipet, Sterile    -   5 mL Serological Pipet, Sterile    -   MF75 Series, Disposable Tissue Culture Filter, 1000 mL, aPES        Filter, 0.2 μm, Sterile    -   Pipets, Serological 100 mL    -   2-mercaptoethanol 1000×, liquid, 55 mM in D-PBS    -   Hank's Balanced Sodium Salt Solution (1×), Liquid, w/o Calcium        Chloride, Magnesium Chloride, Magnesium Sulfate    -   GlutaMAX 1-200 mM (100×), liquid    -   ART Barrier Pipet Tips, 1000 μL, Individually Wrapped, Sterile    -   150 mm Petri Dish, Extra-Depth, Sterile    -   6-well, Ultra-Low Attachment Plates, 9.5 cm² Well Growth Area,        PS, Sterile    -   Thermo Scientific Samco General-Purpose Transfer Pipettes. 7.7        mL, Sterile    -   Repeater Pump Fluid Transfer Set Male Luer Lock End    -   Long Forceps 8″, Sterile    -   Gentamicin Sulfate, 50 mg/mL stock    -   Scientific Disposable Forceps, 4.5″, Stainless Steel, Sterile    -   100 mm petri dish, Sterile Extra Depth    -   Pumpmatic Liquid-Dispensing System    -   Gentamicin Sulfate, 50 mg/mL stock    -   Syringe Cap Dual Function, Red    -   RPMI-1640, 1 L Bottle    -   G-Rex 100M Flask Closed System    -   Sterile rulers    -   Reconstituted IL-2    -   Human Tumor Sample, Head and Neck □N/A    -   Human Tumor Sample, Cervical □N/A    -   GemCell Human Serum AB, Heat Inactivated □N/A    -   Human Tumor Sample, Melanoma    -   GemCell Human Serum AB, Heat Inactivated

Materials: CM2 Preparation/Day 11 REP Seed

-   -   Luer-Lok Syringe, 60 mL Sterile Needle 16G×1.5″ Sterile    -   50 mL Serological Pipets, Sterile    -   1 mL Serological Plastic, Pipet, Sterile    -   Nunc Internally Threaded Cryotube Vials, Sterile    -   10 mL Serological Pipet, Sterile    -   Centrifuge Tube, 15 mL    -   Centrifuge Tube, 50 mL    -   Pipets, Serological 100 mL    -   Syringe, 1 cc Sterile Luer-Lok    -   3 mL Syringe, Luer-Lok Tip, Sterile    -   5 mL Serological Pipet, Sterile    -   Nalgene *MF75* Series Filter Unit Receiver, 250 mL, Sterile    -   Nalgene MF75 Series Filter Unit Receiver, 500 mL, Sterile    -   1000 mL Nalgene Rapid-Flow Sterile Disposable Filter Unit, 0.22        μm PES    -   CryoStor CS-10    -   2-mercaptoethanol 1000× Liquid, 55 mM in D-PBS    -   GlutaMAX 1-200 mM (100×), liquid    -   1,000 μL ART Barrier Sterile Pipet Tips, Individual Wrap    -   VIA1 Cassettes    -   Transfer Pack Container, 1000 mL w/Coupler, Sterile    -   Transfer Pack 300 mL w/Coupler    -   Sterile Alcohol Pads    -   Repeater Pump Fluid Transfer Set Male Luer Lock Ends    -   CTS AIM V 1 L Bottle    -   MACS GMP CD3 pure (OKT-3)    -   Gentamicin Sulfate, 50 mg/mL stock    -   4″ Tubing w/Piercing Pin and Syringe Adapter    -   Syringe Only Luer-Lok 10 mL    -   Tubing, Four Spike Male Luer Manifold    -   Gravity Blood Administration Set Y-type with No injection site,        170 μm blood filter    -   Pumpmatic Liquid-Dispensing System    -   10 L Labtainer 3 Port Bag    -   Gentamicin Sulfate, 50 mg/mL stock    -   100 mL Syringe    -   3000 mL Culture Bag    -   Origen Cell Connect CC2    -   Syringe Cap Dual Function Red    -   RPMI-1640, 1L Bottle    -   G-Rex 500M Flask Closed System    -   Reconstituted IL-2    -   Allogeneic Irradiated Feeder Cells    -   Allogeneic Irradiated Feeder Cells    -   Human Serum, type AB(HI) Gemini    -   Human Serum, type AB(HI) Gemini

Materials: CM4 Preparation/Day 16

-   -   Luer-Lok Syringe, 60 mL Sterile    -   1 mL Serological Plastic Pipet, Sterile    -   Nunc Internally Threaded Cyrotube Vials, Sterile    -   10 mL Serological Pipets, Sterile    -   Centrifuge Tube, 15 mL    -   Centrifuge Tube, 50 mL    -   Pipets, Serological 100 mL    -   Syringe with Luer-Lock, sterile, 3 mL    -   5 mL Serological Pipet, Sterile    -   Syringe only Luer-Lok 10 mL    -   Nalgene *MF75* Series    -   Filter Unit Receiver, 250 mL, Sterile    -   GlutaMAX1-200 mM (100×), liquid    -   ART Barrier Pipet Tips, 1000 μL, Individually Wrapped, Sterile    -   VIA1 Cassettes

Materials: CM4 Preparation/Day 16

-   -   Transfer Pack Container, 1000 mL with Coupler, Sterile    -   Sterile Alcohol Pads    -   Repeater Pump Fluid Transfer Set Male Luer Lock Ends    -   CTS AIM-V 1000 mL □N/A    -   Plasma Transfer Set 4″ Tubing with Female Luer Adapter    -   30 mL Luer-Lok Sterile Syringe    -   CTS AIM V 10 L bag    -   Pumpmatic Liquid-Dispensing System    -   10 L Labtainer 3 Port Bag    -   Syringe Cap Dual Function Red    -   G-Rex500M Flask Closed System    -   Reconstituted IL-2

Materials: Day 22 Formulation, Fill, Cryopreservation

-   -   Luer-Lok Syringe, 60 mL Sterile    -   Needle 16G×1.5″ Sterile    -   50 mL Serological Pipets, Sterile    -   Nunc Internally Threaded Cryotubes Vials, Sterile    -   10 mL Serological Pipet, Sterile    -   Centrifuge Tube, 15 mL    -   Centrifuge Tube, 50 mL    -   Syringe, 1 cc Sterile Luer-Lok    -   3 mL Syringe, Luer-Lok Tip, Sterile    -   25 mL Serological Pipet, Sterile    -   5 mL Serological Pipet, Sterile    -   Syringe only Luer-Lok 10 mL    -   Pipets, Serological 100 mL    -   ART Barrier Sterile Pipet Tips, 200 μL Individual Wrap    -   VIA1-Cassettes    -   Plasma-Lyte A Injection 1 L    -   LOVO Cell Washing Disposable Set    -   LOVO Ancillary Bag Kit    -   Sterile Alcohol Pads    -   Repeater Pump Fluid Transfer Set Male Luer Lock Ends    -   CTS AIM V 1 L Bottle    -   Human Albumin 25%    -   Plasma Transfer Set 4″ Tubing with Female Luer Adapter    -   Tubing, Four Male Luer Manifold    -   Gravity Blood Administration    -   Set Y-type with No injection site, 170 μm blood filter    -   Pumpmatic Liquid-Dispensing System    -   10 L Labtainer 3 Port Bag    -   100 mL Syringe    -   Cryo bag CS750    -   3 L Culture Bag    -   Origen Cell Connect CC2    -   Syringe Cap Dual Function Red    -   Cryostor CS10, 100 mL Bag    -   Dispensing Spike, Vented    -   Reconstituted IL-2

Process

8.1 Day 0 CM1 Media Preparation

-   -   8.1.1 Checked room sanitization, line clearance, and materials.        Confirmed room sanitization,    -   8.1.2 Ensured completion of pre-processing table.    -   8.1.3 Environmental Monitoring. Prior to processing, ensured        pre-process environmental monitoring had been initiated.    -   8.1.4 Prepared RPMI 1640 Media In the BSC, using an        appropriately sized pipette, removed 100.0 mL from 1000 mL RPMI        1640 Media and placed into an appropriately sized container        labeled “Waste”.    -   8.1.5 In the BSC added reagents to RPMI 1640 Media bottle. Added        the following reagents to the RPMI 1640 Media bottle as shown in        in table. Recorded volumes added.    -   Amount Added per bottle: Heat Inactivated Human AB Serum (100.0        mL); GlutaMax (10.0 mL); Gentamicin sulfate, 50 mg/mL (1.0 mL);        2-mercaptoethanol (1.0 mL)    -   8.1.6 Mixed Media. Capped RPMI 1640 Media bottle from Step 8.1.5        and swirled bottle to ensure reagents were mixed thoroughly.    -   8.1.7 Filtered RPMI media. Filtered RPMI 1640 Media from Step        8.1.6 through 1 L 0.22-micron filter unit.    -   8.1.8 Labeled filtered media. Aseptically capped the filtered        media and labeled with the following information.    -   8.1.9 Removed unnecessary materials from BSC. Passed out media        reagents from BSC, left Gentamicin Sulfate and HBSS in BSC for        Formulated Wash Media preparation in Section 8.2.    -   8.1.10 Stored unused consumables. Transferred any remaining        opened/thawed media reagents to appropriate storage conditions        or disposed into waste.    -   NOTE: Assigned the appropriate open expiry to media reagents per        Process Note 5.9 and labeled with batch record lot number    -   8.1.11 Thawed IL-2 aliquot. Thawed one 1.1 mL IL-2 aliquot        (6×10⁶ IU/mL) (BR71424) until all ice had melted. Recorded IL-2:        Lot # and Expiry (NOTE: Ensured IL-2 label was attached).    -   8.1.12 Transferred IL-2 stock solution to media. In the BSC,        transferred 1.0 mL of IL-2 stock solution to the CM1 Day 0 Media        Bottle prepared in Step 8.1.8. Added CM1 Day 0 Media 1 bottle        and IL-2 (6×10⁶ IU/mL) 1.0 mL.    -   8.1.13 Mixed and Relabeled. Capped and swirled the bottle to mix        media containing IL-2. Relabeled as “Complete CM1 Day 0 Media”        and assigned new lot number.    -   8.1.14 Sample Media per Sample Plan. Removed 20.0 mL of media        using an appropriately sized pipette and dispensed into a 50 mL        conical tube.    -   8.1.15 Labeled and stored. Sample labeled with sample plan        inventory label and stored “Media Retain” sample at 2-8° C.        until submitted to Login for testing per Sample Plan.    -   8.1.16 Signed for Sampling. Ensured that LIMS sample plan sheet        was completed for removal of the sample.    -   8.1.17 Prepared “Tissue Pieces” conical tube. In BSC,        transferred 25.0 mL of “Complete CM1 Day 0 Media” (prepared in        Step 8.1.13) to a 50 mL conical tube. Labeled the tube as        “Tissue Pieces” and batch record lot number.    -   8.1.18 Passed G-Rex100MCS into BSC. Aseptically passed        G-Rex100MCS (W3013130) into the BSC.    -   8.1.19 Prepared G-Rex100MCS. In the BSC, closed all clamps on        the G-Rex100MCS, leaving vent filter clamp open.    -   8.1.20 Prepared G-Rex100MCS. Connected the red line of        G-Rex100MCS flask to the larger diameter end of the repeater        pump fluid transfer set (W3009497) via luer connection.    -   8.1.21 Prepared Baxa Pump. Staged Baxa pump next to BSC. Removed        pump tubing section of repeater pump fluid transfer set from BSC        and installed in repeater pump.    -   8.1.22 Prepared to pump media. Within the BSC, removed the        syringe from Pumpmatic Liquid-Dispensing System (PLDS)        (W3012720) and discarded.    -   NOTE: Ensured to not compromise the sterility of the PLDS        pipette.    -   8.1.23 Prepared to pump media. Connected PLDS pipette to the        smaller diameter end of repeater pump fluid transfer set via        luer connection and placed pipette tip in “Complete CM1 Day 0        Media” (prepared in Step 8.1.13) for aspiration.    -   Opened all clamps between media and G-Rex100MCS.    -   8.1.24 Pumped Complete CM1 media into G-Rex100MCS flask. Set the        pump speed to “High” and “9” and pumped all Complete CM1 Day 0        Media into G-Rex100MCS flask. Once all media was transferred,        cleared the line and stopped pump.    -   8.1.25 Disconnected pump from flask. Ensured all clamps were        closed on the flask, except vent filter. Removed the repeater        pump fluid transfer set from the red media line, and placed a        red cap (W3012845) on the red media line.    -   8.1.26 Heated seal. Removed G-Rex100MCS flask from BSC, heated        seal (per Process Note 5.12) off the red cap from the red line        near the terminal luer.    -   8.1.27 Labeled G-Rex100MCS. Labeled G-Rex100MCS flask with QA        provided in-process “Day 0” label. Attached sample “Day 0” label        below.    -   8.1.28 Monitored Incubator. Incubator parameters: Temperature        LED Display: 37.0±2.0° C.; CO2 Percentage: 5.0±1.5% CO2    -   8.1.29 Warmed Media. Placed the 50 mL conical tube labeled        “Tissue Fragments” prepared in Step 8.1.17 and the G-Rex100MCS        prepared in Step 8.1.27 in incubator for ≥30 minutes of warming.

Recorded warming times below. Recorded if Warm Time was ≥30 minutes(Yes/No).

[Tissue Fragments Conical or GRex100MCS]

-   -   8.1.30 Reviewed Section 8.1.

8.2 Day 0 Tumor Wash Media Preparation

-   -   8.2.1 Added Gentamicin to HBSS. In the BSC, added 5.0 mL        Gentamicin (W3009832 or W3012735) to 1×500 mL HBSS Media        (W3013128) bottle. Recorded volumes. Added per bottle: HBSS        (500.0 mL); Gentamicin sulfate, 50 mg/ml (5.0 mL)    -   8.2.2 Capped HBSS bottle and swirled. Capped HBSS containing        gentamicin prepared in Step 8.2.1 and swirled bottle to ensure        reagents are mixed thoroughly.    -   8.2.3 Filtered Solution. Filtered HBSS containing gentamicin        prepared in Step 8.2.1 through a 1 L 0.22-micron filter unit        (W1218810).    -   8.2.4 Aseptically capped the filtered media and label.        Aseptically capped the filtered media and labeled with the        following information. Proceeded to SECTION 8.3.    -   8.2.5 Reviewed Section 8.2.

8.3 Day 0 Tumor Processing

-   -   8.3.1 Obtained Tumor. Obtained tumor specimen from QAR and        transferred into suite at 2-8° C. immediately for processing.        Ensured all necessary information is recorded on the Tumor        Shipping Batch Record.    -   8.3.2 Recorded Tumor Information.    -   8.3.3 Affixed Tumor Label. Affixed tumor Attachment. QAR release        sticker below. Attached Tumor Shipping Batch Record as #5.    -   8.3.4 Passed in necessary materials for tumor dissection into        the BSC.    -   8.3.5 Opened Materials. Opened all materials inside the BSC,        ensuring not to compromise the sterility of the items.    -   8.3.6 Labeled Materials. Labeled three 50 ml conical tubes: the        first as “Forceps,” the second as “Scalpel,” and the third as        “Fresh Tumor Wash Media”. Labeled 5×100 mm petri dishes as “Wash        1,” “Wash 2,” “Wash 3,” “Holding,” and “Unfavorable.” Labeled        one 6 well plate as “Favorable Intermediate Fragments.”    -   8.3.7 Aliquoted Tumor Wash Media. Using an appropriately sized        pipette, transferred 5.0 mL of “Tumor Wash Media” prepared in        Step 8.2.4 into each well of one 6-well plate for favorable        intermediate tumor fragments (30.0 mL total). NOTE: The forceps        and scalpels were stored in their respective tumor wash media        conicals as needed during the tumor washing and dissection        processes.    -   8.3.8 Aliquoted Tumor Wash Media. Using an appropriately sized        pipette, transferred 50.0 mL of “Tumor Wash Media” prepared in        Step 8.2.4 into each 100 mm petri dish for “Wash 1,” “Wash 2,”        “Wash 3,” and “Holding” (200.0 mL total).    -   8.3.9 Aliquoted Tumor Wash Media. Using an appropriately sized        pipette, transfer 20.0 mL of “Tumor Wash Media” prepared in Step        8.2.4 into each 50 mL conical (60.0 mL total).    -   8.3.10 Prepared Lids for Tumor Pieces. Aseptically removed lids        from two 6-well plates. The lids were utilized for selected        tumor pieces. NOTE: Throughout tumor processing, DID NOT cross        over open tissue culture plates and lids.    -   8.3.11 Passed the tumor into the BSC. Aseptically passed the        tumor into the BSC. Recorded processing start time.    -   8.3.12 Tumor Wash 1 Using 8″ forceps (W3009771), removed the        tumor from the specimen bottle and transferred to the “Wash 1”        dish prepared in Step 8.3.8.    -   NOTE: Retained the solution in specimen bottle.    -   8.3.13 Tumor Wash 1 Using forceps, gently washed tumor time from        timer below: specimen and allowed it to sit for ≥3 minutes.        Recorded wash time (MM:SS).    -   8.3.14 Prepared Bioburden Sample per Sample Plan. Transferred        20.0 mL (or available volume) of solution from the tumor        specimen bottle into a 50 mL conical per sample plan.    -   8.3.15 Labeled and stored sample. Labeled with sample plan        inventory label and stored bioburden sample collected in Step        8.3.14 at 2-8° C. until submitted for testing.    -   8.3.16 Signed for sampling. Ensured that LIMS sample plan sheet        was completed for removal of the sample.    -   8.3.17 Tumor Wash 2. Using a new set of forceps removed the        tumor from the “Wash 1” dish and transferred to the “Wash 2”        dish prepared in Step 8.3.8. 8.3.18 Tumor Wash 2. Using forceps,        washed tumor specimen by gently agitating for ≥3 minutes and        allowed it to sit. Recorded time.    -   8.3.19 Prepared drops of Tumor Wash Media for desired tumor        pieces. Using a transfer pipette, placed 4 individual drops of        Tumor Wash Media from the conical prepared in Step 8.3.9 into        each of the 6 circles on the upturned lids of the 6-well plates        (2 lids). Placed an extra drop on two circles for a total of 50        drops.    -   8.3.20 Tumor Wash 3. Using forceps, removed the tumor from the        “Wash 2” dish and transferred to the “Wash 3” dish prepared in        Step 8.3.8.    -   8.3.21 Tumor Wash 3. Using forceps, washed tumor specimen by        gently agitating and allowed it to sit for ≥3 minutes. Recorded        time.    -   8.3.22 Prepared tumor dissection dish. Placed a ruler under 150        mm dish lid.    -   8.3.23 Transferred Tumor to Dissection Dish. Using forceps,        aseptically transferred tumor specimen to the 150 mm dissection        dish lid.    -   8.3.24 Measured Tumor. Arranged all pieces of tumor specimen end        to end and recorded the approximate overall length and number of        fragments. Took a clear picture of each tumor specimen.    -   8.3.25 Assessed Tumor. Assessed the tumor for necrotic/fatty        tissue. Assessed whether >30% of entire tumor area observed to        be necrotic and/or fatty tissue; if yes, contacted area        management to ensure tumor was of appropriate size, then        proceeded to Step 8.3.26. Assessed whether <30% of entire tumor        area were observed to be necrotic or fatty tissue; if yes,        proceeded to Step 8.3.27 and clean-up dissection was NOT        performed.    -   8.3.26 If applicable: Clean-Up Dissection. If tumor was large        and >30% of tissue exterior was observed to be necrotic/fatty,        performed “clean up dissection” by removing necrotic/fatty        tissue while preserving tumor inner structure using a        combination of scalpel and/or forceps. NOTE: To maintain tumor        internal structure, used only vertical cutting pressure. Did not        cut in a sawing motion with scalpel. NOTE: Fat, necrotic, and        extraneous tissue were placed in unfavorable dish.    -   8.3.27 Dissect TumorUsing a combination of scalpel and/or        forceps, cut the tumor specimen into even, appropriately sized        fragments (up to 6 intermediate fragments). NOTE: To maintain        tumor internal structure, use only vertical cutting pressure.        Did not cut in a sawing motion with scalpel. NOTE: Ensured to        keep non-dissected intermediate fragments completely submerged        in “Tumor Wash Media” (prepared in Step 8.2.4).    -   8.3.28 Transferred intermediate tumor fragments. Transferred        each intermediate fragment to the “holding” dish from Step        8.3.8.    -   8.3.29 Dissected Tumor Fragments. Manipulated one intermediate        fragment at a time, dissected the tumor intermediate fragment in        the dissection dish into pieces approximately 3×3×3 mm in size,        minimizing the amount of hemorrhagic, necrotic, and/or fatty        tissues on each piece. NOTE: To maintain tumor internal        structure, used only vertical cutting pressure. Did not cut in a        sawing motion with scalpel.    -   8.3.30 Selected Tumor Pieces. Selected up to eight (8) tumor        pieces without hemorrhagic, necrotic, and/or fatty tissue. Used        the ruler for reference. Continued dissection until 8 favorable        pieces have been obtained, or the entire intermediate fragment        has been dissected. Transferred each selected piece to one of        the drops of “Tumor Wash Media” prepared in Step 8.3.19.    -   8.3.31 Stored Intermediate Fragments to Prevent Drying. After        selecting up to eight (8) pieces from the intermediate fragment,        placed remnants of intermediate fragment into a new single well        of “Favorable Intermediate Fragments” 6-well plate prepared in        Step 8.3.7. NOTE: Fatty or necrotic tissue was placed in the        “Unfavorable” dish (prepared in step 8.3.6).    -   8.3.32 Repeated Intermediate Fragment Dissection. Proceeded to        the next intermediate fragment, repeated Steps 8.3.29-8.3.31        until all intermediate fragments had been processed, obtained        fresh scalpels and forceps as needed.    -   8.3.33 Determined number of pieces collected. If desirable        tissue remains, selected additional Favorable Tumor Pieces from        the “favorable intermediate fragments” 6-well plate to fill the        drops for a maximum of 50 pieces. Recorded the total number of        dissected pieces created. NOTE: Ensuring to keep the tumor        intermediate fragments hydrated with Wash Medium as necessary        throughout dissection. Recorded Total quantity of dissected        pieces collected.    -   8.3.34 Removed Conical Tube from Incubator. Removed the “Tissue        Pieces” 50 mL conical tube from the incubator. Recorded time in        Step 8.1.29. Ensured conical tube was warmed for ≥30 min.    -   8.3.35 Prepared Conical Tube. Passed “Tissue Pieces” 50 mL        conical into the BSC, ensuring not to compromise the sterility        of open processing surfaces.    -   8.3.36 Transferred Tumor Pieces to 50 mL Conical Tube. Using a        transfer pipette, scalpel, forceps or combination, transferred        the selected 50 best tumor fragments from favorable dish lids to        the “Tissue Pieces” 50 mL conical tube.    -   NOTE: If a tumor piece was dropped during transfer and desirable        tissue remains, additional pieces from the favorable tumor        intermediate fragment wells were added. Recorded numbers of        pieces.    -   8.3.37 Prepared BSC for G-REX100MCS. Removed all unnecessary        items from BSC for vessel seed, retaining the favorable tissue        plates if they contained extra fragments.    -   8.3.38 Removed G-REX100MCS from Incubator. Removed G-Rex100MCS        containing media from incubator. Completed Step 8.1.29.    -   8.3.39 Passed flask into BSC. Aseptically passed G-Rex100MCS        flask into the BSC. NOTE: When transferring the flask, did not        hold from the lid or the bottom of the vessel. Transferred the        vessel by handling the sides. NOTE: Only utilized IPA WIPES when        handling G-Rex flasks.    -   8.3.40 Added tumor fragments to G-Rex100MCS flask. In the BSC,        lifted G-Rex100MCS flask cap, ensuring that sterility of        internal tubing was maintained.    -   Swirled conical tube with tumor pieces to suspend and quickly        poured the contents into the G-Rex100MCS flask.    -   8.3.41 Evenly distributed pieces. Ensured that the tumor pieces        were evenly distributed across the membrane of the flask. Gently        tilted the flask back and forth if necessary to evenly        distribute the tumor pieces.    -   8.3.42 Recorded total number of tumor fragments in vessel.        Recorded number of tumor fragments on bottom membrane of vessel        and number of observed to be floating in vessel. NOTE: If the        number of fragments seeded were NOT equivalent to number of        collected in Step 8.3.36H, contacted Area Management, and        document in Section 10.0.    -   8.3.43 Incubate G-Rex flask Incubated G-Rex100MCS at the        following parameters: Incubated G-Rex flask: Temperature LED        Display: 37.0±2.0° C.; CO2 Percentage: 5.0±1.5% CO2    -   8.3.44 Calculated incubation window. Performed calculations to        determine the proper time to remove G-Rex100MCS incubator on        Day 11. Calculations: Time of incubation; lower limit=time of        incubation+252 hours; upper limit=time of incubation+276 hours    -   8.3.45 Environmental Monitoring. After processing, verified BSC        and personnel monitoring were performed.    -   8.3.46 Discarded materials. Stored remaining unwarmed media at        2-8° C. and labeled. After process was complete, discarded any        remaining warmed media and thawed aliquots of IL-2.    -   8.3.47 Sample submission. Ensured all Day 0 samples were        submitted to Login and transferred in LIMS.    -   8.3.48 Review Section 8.3.

8.4 Day 11—Media Preparation

-   -   8.4.1 Checked room, sanitization, line clearance, and materials.        Confirmed room sanitization, line clearance, and that materials        are within expiry.    -   8.4.2 Pre-processing table. Equipment list: BSC; Balance; Sebra        Tube Sealer; Gatherex™ Media Removal and Cell Recovery Device;        Ensure QA provided placard is placed on the appropriate BSC;        Ensure QA provided placard lot number and patient ID display        matches the lot number and patient ID in this Batch Record.    -   8.4.3 Monitored Incubator. Monitored Incubator. Incubator        parameters: Temperature LED Display: 37.0±2.0° C.; CO2        Percentage: 5.0±1.5% CO2.    -   NOTE: Section 8.4 may be run concurrently with section 8.5.        8.4.4 Warmed media. Warmed 3×1000 mL RPMI 1640 Media (W3013112)        bottles and 3×1000 mL AIM-V (W3009501) bottles in an incubator        for ≥30 minutes. Recorded time. Media: RPMI 1640 and AIM-V.    -   NOTE: Placed an additional 1×1000 ml bottle of AIM-V Media        (W3009501) at room temperature for use in Step 8.5.34. Labeled        the bottle “For Cell Count Dilutions Only” and the batch record        lot number.    -   8.4.5 Environmental monitoring. Prior to processing, ensured        pre-process environmental monitoring was performed as per        SOP-00344.    -   8.4.6 Removed RPMI 1640 Media from incubator. Removed the RPMI        1640 Media when time was reached. Record end incubation time in        Step    -   8.4.4. Ensure media was warmed for ≥30 min.    -   8.4.7 Prepared RPMI 1640 Media. In the BSC, removed 100.0 mL        from each of the three pre-warmed 1000 mL RPMI 1640 Media        bottles and placed into an appropriately sized container labeled        “Waste”.    -   8.4.8 In BSC add reagents to RPMI 1640 Media bottle. In the BSC        added the following reagents to each of the three RPMI 1640        Media bottles.

Recorded volumes added to each bottle. GemCell Human serum, HeatInactivated Type AB (100.0 mL), GlutaMax (10.0 mL), Gentamicin sulfate,50 mg/ml (1.0 mL), 2-mercaptoethanol (1.0 mL)

-   -   8.4.9 Filter Media. Caped bottles from Step 8.4.8 and swirled to        ensure reagents were mixed thoroughly. Filtered each bottle of        media through a separate 1 L 0.22-micron filter unit.    -   8.4.10 Labeled filtered media. Aseptically capped the filtered        media and labeled each bottle with CM1 Day 11 Media.    -   8.4.11 Thawed IL-2 aliquot. Thawed 3×1.1 mL aliquots of IL-2        (6×106 IU/mL) (BR71424) until all ice had melted Recorded IL 2        lot # and Expiry.    -   NOTE: EnsureIL-2 label is attached.    -   8.4.12 Removed AIM-V Media from the incubator. Removed the three        bottles of AIM-V Media from the incubator. Recorded end        incubation time in Step    -   8.4.4. Ensured media had been warmed for ≥30 minutes.    -   8.4.13 Add IL-2 to AIM-V. In the BSC, using a micropipette,        added 3.0 mL of thawed IL-2 into one 1 L bottle of pre-warmed        AIM-V media. Rinse micropipette tip with media after dispensing        IL-2. Use a new sterile micropipette tip for each aliquot.        Recorded the total volume added. Labeled bottle as “AIM-V        Containing IL-2”.    -   8.4.14 Transferred materials. Aseptically transferred a 10 L        Labtainer Bag and a repeater pump transfer set into the BSC.    -   8.4.15 Prepared 10 L Labtainer media bag. Closed all lines on a        10 L Labtainer bag. Attached the larger diameter tubing end of a        repeater pump transfer set to the middle female port of the 10 L        Labtainer Bag via luer lock connection.    -   8.4.16 Prepare Baxa pump. Staged the Baxa pump next to the BSC.        Fed the transfer set tubing through the Baxa pump situated        outside of the BSC.    -   Set the Baxa Pump to “High” and “9”.    -   8.4.17 Prepared 10 L Labtainer media bag. In BSC, removed        syringe from Pumpmatic Liquid-Dispensing System (PLDS) and        discarded. NOTE: Ensured to not compromise the sterility of the        PLDS pipette.    -   8.4.18 Prepared 10 L Labtainer media bag. Connected PLDS pipette        to smaller diameter end of repeater pump fluid transfer set via        luer connection and placed pipette tip in AIM-V media containing        IL-2 bottle (prepared in Step 8.4.13) for aspiration. Opened all        clamps between media bottle and 10 L Labtainer.    -   8.4.19 Pumped media into 10 L Labtainer. In the BSC, using the        PLDS, transfer pre-warmed AIM-V media containing IL-2 prepared        in Step 8.4.13, as well as two additional AIM-V bottles into the        10 L Labtainer bag. Added the three bottles of filtered CM1 Day        11 Media from Step 8.4.10. After addition of final bottle,        cleared the line to the bag. NOTE: Stopped the pump between        addition of each bottle of media.    -   8.4.20 Removed pumpmatic from Labtainer bag. Removed PLDS from        the transfer set and placed a red cap on the luer of the line in        the BSC.    -   8.4.21 Mixed media. Gently massaged the bag to mix.    -   8.4.22 Labeled media. In the BSC, labeled the media bag with the        following information. Expiration date was 24 hours from the        preparation date.    -   8.4.23 Sample media per sample plan. In the BSC, attached a 60        mL syringe to the available female port of the “Complete CM2 Day        11 Media” bag prepared in step 8.4.22. Removed 20.0 mL of media        and place in a 50 mL conical tube.    -   Placed a red cap on the female port of the “Complete CM2 Day 11        Media” Bag.    -   8.4.24 Labeled and stored sample. Labeled with sample plan        inventory label and stored Media Retain Sample at 2-8° C. until        submitted to Login for testing.    -   8.4.25 Sign for Sampling. Ensured that LIMS sample plan sheet        was completed for removal of the sample.    -   8.4.26 Sealed the transfer set line. Outside the BSC, heat        sealed off (per Process Note 5.12) the red cap on the transfer        set line, close to red cap. Kept the transfer set on the bag.    -   8.4.27 Prepared Cell Count Dilution Tubes In the BSC, added 4.5        mL of AIM-V Media that had been labelled with “For Cell Count        Dilutions” and lot number to four 15 mL conical tubes. Labeled        the tubes with the lot number and tube number (1-4). Labeled 4        cryovials “Feeder” and vial number (1-4). Kept vials under BSC        to be used in Step 8.5.30.    -   8.4.28 Transferred reagents from the BSC to 2-8° C. Transferred        any remaining 2-mercaptoethanol, GlutaMax, and human serum from        the BSC to 2-8° C. Ensured all reagents were labeled with the        batch record lot number, and the appropriate open expiry per        Process Note 5.9.    -   8.4.29 Prepared 1 L Transfer Pack. Outside of the BSC weld (per        Process Note 5.11) a 1 L Transfer Pack to the transfer set        attached to the “Complete CM2 Day 11 Media” bag prepared in step        8.4.22. Labeled transfer pack as “Feeder Cell CM2 Media” and lot        number.    -   8.4.30 Prepared 1 L Transfer Pack. Made a mark on the tubing of        the 1 L Transfer Pack tubing a few inches away from the bag.        Placed the empty Transfer Pack onto the scale so that the tubing        was on the scale to the point of the mark.    -   8.4.31 Tared scale. Tared the scale and left the empty Transfer        Pack on the scale.    -   8.4.32 Prepared feeder cell transfer pack. Set the Baxa pump to        “Medium” and “4.” Pumped 500.0±5.0 mL of “Complete CM2 Day 11”        media prepared in Step 8.4.22 into Cell CM2 Media” transfer        pack. Measured by weight and recorded the volume of Complete CM2        media added to the Transfer Pack.    -   8.4.33 Heated seal line. Once filled, heated seal the line per        Process Note 5.12. Separated CM2 Day 11 media bag with transfer        set from feeder cell media transfer pack, kept weld toward 1 L        transfer pack.    -   8.4.34 If applicable: Incubated feeder cell media transfer pack.        When applicable, placed the “Feeder Cell CM2 Media” transfer        pack in incubator until used in Step 8.6.6.    -   8.4.35 Incubated Complete CM2 Day 11 Media. Placed “Complete CM2        Day 11 Media” prepared in Step 8.4.22 in incubator until use in        Step 8.7.2. 8.4.36 Reviewed Section 8.4.

8.5 Day 11—TIL Harvest

-   -   8.5.1 Preprocessing table. Monitored incubator. Incubator        parameters: Temperature LED Display: 37.0±2.0° C.; CO2        Percentage: 5.0±1.5% CO2.    -   NOTE: Section 8.5 may be run concurrently with Sections 8.4 and        8.6.    -   8.5.2 Removed G-Rex100MCS from incubator. Performed check below        to ensure incubation parameters are met before removing        G-Rex100MCS from incubator. Lower limit from Step 8.3.44 B.        Upper limit from Step 8.3.44 C. Record Time of Removal from        incubator. Determined: Is 8.3.44 B≤Time of Removal from        incubator <Step 8.3.44 C? *IF NO CONTACT AREA MANAGEMENT.        Carefully removed G-Rex100MCS from incubator and ensured all        clamps were closed except large filter line. Recorded processing        start time.    -   8.5.3 Prepared 300 mL Transfer Pack. Labeled a 300 mL Transfer        pack as “TIL Suspension”.    -   8.5.4 Prepared 300 mL Transfer Pack. Sterile welded (per Process        Note 5.11) the TIL Suspension transfer (single line) of a        Gravity Blood Filter. See, for example.    -   8.5.5 Prepared 300 mL Transfer Pack. Placed the 300 mL Transfer        Pack on a scale and record dry weight.    -   8.5.6 Prepared 1 L Transfer Pack. Labeled 1 L Transfer Pack as        “Supernatant” and Lot number.    -   8.5.7 Welded transfer packs to G-Rex100MCS. Sterile welded (per        Process Note 5.11) the red media removal line from the        G-Rex100MCS to the “Supernatant” transfer pack. Sterile welded        the clear cell removal line from the G-Rex100MCS to one of the        two spike lines on the top of the blood filter connected to the        “TIL Suspension” transfer pack prepared in Step 8.5.4. See, for        example.    -   8.5.8 GatheRex Setup. Placed G-Rex100MCS on the left side of the        GatheRex and the “Supernatant” and “TIL Suspension” transfer        packs to the right side.    -   8.5.9 GatheRex Setup. Install the red media removal line from        the G Rex100MCS to the top clamp (marked with a red line) and        tubing guides on the GatheRex. Installed the clear harvest line        from the G-Rex100MCS to the bottom clamp (marked with a blue        line) and tubing guides on the GatheRex.    -   8.5.10 GatheRex Setup. Attached the gas line from the GatheRex        to the sterile filter of the G-Rex100MCS flask. NOTE: Before        removing the supernatant from the G-Rex100MCS flask, ensured all        clamps on the cell removal lines were closed.    -   8.5.11 Volume Reduction of G-Rex100MCS. Transferred ˜900 mL of        culture supernatant from the G-Rex100MCS to the 1 L Transfer        Pack. Visually inspect G-Rex100MCS flask to ensure flask is        level and media has been reduced to the end of the aspirating        dip tube. NOTE: If the Gatherex stops prematurely, it was        restarted by pressing the button with the arrow pointing to the        right again.    -   8.5.12 Prepare flask for TIL Harvest. After removal of the        supernatant, closed all clamps to the red line.    -   8.5.13 Initiation of TIL Harvest. Recorded the start time of the        TIL harvest.    -   8.5.14 Initiation of TIL Harvest. Vigorously tapped flask and        swirled media to release cells. Performed an inspection of the        flask to ensure all cells have detached. NOTE: Contacted area        management if cells did not detach.    -   8.5.15 Initiation of TIL Harvest. Tilt flask away from        collection tubing and allowed tumor pieces to settle along edge.        Slowly tipped flask toward collection tubing so pieces remained        on the opposite side of the flask. NOTE: If the cell collection        straw is not at the junction of the wall and bottom membrane,        rapping the flask while tilted at a 450 angle is usually        sufficient to properly position the straw.    -   8.5.16 TIL Harvested. Released all clamps leading to the TIL        Suspension transfer pack.    -   8.5.17 TIL Harvested. Using the GatheRex, transferred the cell        suspension through the blood filter into the 300 mL transfer        pack. NOTE: Be sure to maintain the tilted edge until all cells        and media are collected.    -   8.5.18 TIL Harvested. Inspect membrane for adherent cells.    -   8.5.19 Rinsed flask membrane. Rinsed the bottom of the        G-Rex100MCS. Cover ˜¼ of gas exchange membrane with rinse media.        NOTE: If tumor pieces obstruct the harvest line, pause        collection by pressing the “X” on the cell collection line.        Press the “Release Clamps” button on the Gatherex and pick the        transfer pack up and gently squeeze with increasing pressure        until the fragment is removed. Do not squeeze the bag too hard,        as this may cause the line or bag to rupture. Resume collection        once obstruction has been removed.    -   8.5.20 Closed clamps on G-Rex100MCS. Ensured all clamps are        closed.    -   8.5.21 Heat sealed. Heat sealed (per Process Note 5.12) the TIL        suspension transfer pack as close to the weld as possible so        that the overall tubing length remains approximately the same.    -   8.5.22 Heat sealed. Heat sealed the “Supernatant” transfer pack        per Process Note 5.12. Maintained enough line to weld.    -   8.5.23 Calculated volume of TIL suspension. Recorded weight of        TIL Suspension transfer pack and calculated the volume of cell        suspension.    -   8.5.24 Prepared Supernatant Transfer Pack for Sampling. Welded        (per Process Note 5.11) a 4″ plasma transfer set to        “supernatant” transfer pack, retaining the luer connection on        the 4″ plasma transfer set, and transferred into the BSC.    -   8.5.25 Prepared TIL Suspension Transfer Pack for Sampling.        Welded (per Process Note 5.11) a 4″ plasma transfer set to 300        mL “TIL Suspension” transfer pack, retained the luer connection        on the 4″ plasma transfer set, and transferred into the BSC.    -   8.5.26 Pulled Bac-T Sample. In the BSC, using an appropriately        sized syringe, draw up approximately 20.0 mL of supernatant from        the 1 L “Supernatant” transfer pack and dispense into a sterile        50 mL conical tube labeled “Bac-T.” Keep in BSC for use in Step        8.5.27.    -   8.5.27 Inoculated BacT per Sample Plan. Removed a 1.0 mL sample        from the 50 mL conical labeled BacT prepared in Step 8.5.26        using an appropriately sized syringe and inoculated the        anaerobic bottle. Recorded the time the bottle was inoculated        using the space provided on the bottle label. Repeated the above        for the aerobic bottle. NOTE: This step may be performed out of        sequence.    -   8.5.28 Labeled and stored sample. Labeled with sample plan        inventory label and stored Bac-T sample at room temperature,        protected from light, until submitted to Login for testing per        Sample Plan. NOTE: Did not cover barcode on bottle with label.    -   8.5.29 Signed for Sampling. Ensured that LIMS sample plan sheet        was completed for removal of the sample.    -   8.5.30 TIL Cell Count Samples. Labeled 4 cryovials with vial        number (1-4). Using separate 3 mL syringes, pulled 4×1.0 mL cell        count samples from TIL Suspension Transfer Pack using the luer        connection, and placed in respective cryovials.    -   8.5.31 Closed the luer connection. Placed a red cap (W3012845)        on the line.    -   8.5.32 Incubated TIL. Placed TIL Transfer Pack in incubator        until needed.    -   8.5.33 Perform Cell Counts Perform cell counts and calculations        utilizing NC-200 and Process Note 5.14. Perform initial cell        counts undiluted.    -   8.5.34 Recorded Cell Count sample volumes. NOTE: If no dilution        needed, “Sample [μL]”=200, “Dilution [μL]”=0.    -   8.5.35 Determined Multiplication Factor. Total cell count sample        Volume: 8.5.34A+8.5.34B. Multiplication Factor C÷8.5.34A.    -   8.5.36 Selected protocols and entered multiplication factors.        Ensured the “Viable Cell Count Assay” protocol had been        selected, all multiplication factors, and sample and diluent        volumes had been entered. NOTE: If no dilution needed, enter        “Sample [μL]”=200, “Dilution [μL]”=0    -   8.5.37 Recorded File Name, Viability and Cell Counts from        Nucleoview    -   8.5.38 Determined the Average of Viable Cell Concentration and        Viability of the cell counts performed. Viability        (8.5.37A+8.5.37B)÷2. Viable Cell Concentration        (8.5.37C+8.5.37D)÷2    -   8.5.39 Determined Upper and Lower Limit for counts. Lower Limit:        8.5.38F×0.9. Upper Limit: 8.5.38F×1.1.    -   8.5.40 Were both counts within acceptable limits? Lower Limit:        8.5.37 C and D≥8.5.39G. Upper Limit: 8.5.37 C and D≤8.5.39H. *If        either result was “No” performed second set of counts in steps        8.5.41-8.5.48*.    -   8.5.41 If Applicable: Performed cell counts. Performed cell        counts and calculations in utilizing NC-200 and Process Note        5.14. NOTE: Dilution was adjusted according based off the        expected concentration of cells. Performed    -   8.5.42 If Applicable: Recorded Cell Count sample volumes.    -   8.5.43 If Applicable: Determined Multiplication Factor. Total        cell count sample Volume: 8.5.42A+8.5.42B. Multiplication Factor        C÷8.5.42A D    -   8.5.44 If Applicable: Selected protocols and entered        multiplication factors. Ensured the “Viable Cell Count Assay”        protocol was selected, all multiplication factors, and sample        and diluent volumes were entered. NOTE: If no dilution needed,        enter “Sample [μL]”=200, “Dilution [μL]”=0.    -   8.5.45 If Applicable: Recorded Cell Counts from Nucleoview    -   8.5.46 If Applicable: Determined the Average of Viable Cell        Concentration and Viability of the cell counts performed.        Determined averaged viable cell concentration.    -   8.5.47 If Applicable: Determined Upper and Lower Limit for        counts. Lower Limit: 8.5.46F×0.9. Upper Limit: 8.5.46F×1.1.    -   8.5.48 If Applicable: Were counts within acceptable limits?        Lower Limit: 8.5.45 C and D≥8.5.47G. Upper Limit: 8.5.45 C and        D≤8.5.47H. NOTE: If either result is “No” continue to Step        8.5.49 to determine an average.    -   8.5.49 If Applicable: Determined an average Viable Cell        Concentration from all four counts performed. Average Viable        Cell Concentration (A+B+C+D)÷4=AVERAGE    -   8.5.50 Adjusted Volume of TIL Suspension Calculate the adjusted        volume of TIL suspension after removal of cell count samples.        Total TIL Cell Volume from Step 8.5.23C (A). Volume of Cell        Count Sample Removed (4.0 ml) (B) Adjusted Total TIL Cell Volume        C=A−B.    -   8.5.51 Calculated Total Viable TIL Cells. Average Viable Cell        Concentration*: 8.5.38 F* or 8.5.46 F* or *8.5.49E*; Total        Volume: 8.5.50; Total Viable Cells: C=A×B. *Circle step        reference used to determine Viable Cell Concentration. NOTE: If        Total Viable TIL Cells is <5×10⁶ cells contact Area Management        and proceed to Step 8.7.1. If Total Viable TIL Cells is >5×10⁶,        proceed to Step 8.5.52.    -   8.5.52 Calculation for flow cytometry. If the Total Viable TIL        Cell count from Step 8.5.51C was ≥4.0×10⁷, calculated the volume        to obtain 1.0×10⁷ cells for the flow cytometry sample. *If there        are <4.0×10⁷ cells, N/A the remaining fields in the table.        Proceed to Step 8.7.1. Total viable cells required for flow        cytometry: 1.0×10⁷ cells. Volume of cells required for flow        cytometry: Viable cell concentration from 8.5.51 divided by        1.0×10⁷ cells A.    -   8.5.53 If Applicable: Removed TIL from incubator. Removed TIL        Suspension from incubator and recorded end incubation time in        Step 8.5.32.    -   8.5.54 If Applicable: Removed flow cytometry sample as per        Sample Plan. Using an appropriately sized syringe, removed the        calculated volume (8.5.52 C) for the phenotyping sample from the        TIL Suspension transfer pack and place in a 50 mL conical tube.    -   8.5.55 If Applicable: Labeled and stored flow cytometry sample.        Labeled with sample plan inventory label and store Flow        Cytometry sample at 2-8° C. until submitted to Login for testing        per Sampling Plan.    -   8.5.56 Signed for Sampling. Ensure that LIMS sample plan sheet        was completed for removal of the sample.    -   8.5.57 If Applicable: Recalculated Total Viable Cells and Volume        flow. Calculated the remaining Total Viable Cells and remaining        volume after the removal of cytometry sample below.

Parameter Formula Result Total Viable TIL Step 8.5.51C A. cells TILremoved for Flow 1 × 10⁷ cells B. 1 × 10⁷ cells Cytometry RemainingTotal C = A − B C. cells Viable TIL Volume of TIL Step 8.5.50C D. mLVolume of TIL Step 8.5.52 C E. mL removed Remaining Volume of F = D − EF. mL TIL

-   -   8.5.58 If Applicable: Calculated TIL volume. Calculated the        volume of TIL suspension equal to 2.0×10⁸ viable cells.

Volume of TIL Suspension containing Total Viable Cells Viable CellConcentration 2.0 × 10⁸ viable cells Required from Step 8.5.51A C = A ÷B A. 2.0 × 10⁸ cells B. cells/mL C. mL

-   -   8.5.59 If Applicable: Calculated TIL volume to remove Calculate        the excess volume of TIL cells to remove.

Volume of suspension Total Volume of TIL containing 2.0 × 10⁸ Volume ofexcess TIL to Suspension from Step TIL from Step Remove 8.5.57F 8.5.58CC = A − B A. mL B. mL C. mL

-   -   8.5.60 If Applicable: Removed excess TIL. In the BSC, using an        appropriately sized syringe, remove the calculated volume (Step        8.5.59C) from the TIL Suspension transfer pack. NOTE: Do not use        a syringe more than once. Use multiple syringes if applicable.        Placed in appropriately sized sterile container and label as        date, and lot number. Placed a red cap on the “TIL Suspension”        transfer pack line.    -   8.5.61 If Applicable: Placed TIL in Incubator. Placed TIL        Suspension Transfer Pack in incubator until needed. Recorded        time.    -   8.5.62 If Applicable: Calculations. Calculated total excess TIL        removed.    -   Step 8.5.51A Volume of TIL to Remove from Step 8.5.59C.        Calculated Total Excess TIL removed.

Viable Cell Total Excess TIL Concentration from Volume of TIL to Removeremoved Step 8.5.51A from Step 8.5.59C C = A × B A. cells/mL B. mL C.cells

-   -   8.5.63 If Applicable: Calculations. Calculated amount of CS-10        media to add to excess TIL cells from Step 8.5.62C. Target cell        concentration for freezing is 1.0×10⁸ cells/ml.

Volume of CS-10 to Total Excess TIL Add Removed Target Concentration to(mL) Step 8.5.62C Freeze C = A ÷ B A. cells B. 1.0 × 10⁸ cells/mL C. mL

-   -   8.5.64 If Applicable: Centrifuged excess TIL. Centrifuged the        excess TIL cell suspension. Speed: 350×g. Time: 10:00 minutes.        Temperature: Ambient Brake: Full (9). Acceleration: Full (9).    -   8.5.65 If Applicable: Observed conical tube. Recorded        observations: Pellet observed? Supernatant was clear? *NOTE: If        either answer was no, contact Area Management.    -   8.5.66 If Applicable: Added CS-10. In BSC, aseptically aspirate        supernatant. Gently tap bottom of tube to resuspend cells in        remaining fluid.    -   8.5.67 If Applicable: Added CS10. Slowly add the volume of CS10        calculated in Step 8.5.63C    -   8.5.68 If Applicable: Labeled vials. Labeled vials with QA        provided labels. Attached a sample label.    -   8.5.69 If Applicable: Filled Vials. Aliquoted 1.0 mL cell        suspension, into appropriately sized cryovials. NOTE: Did not        fill more than 10 vials of Excess TIL.    -   8.5.70 If Applicable: Filled Vials. Aliquoted residual volume        into appropriately sized cryovial per SOP-00242. If volume is        <0.5 mL, add CS10 to vial until volume is 0.5 mL.    -   8.5.71 If Applicable: Filled Vials. Filled one vial with 1.0 mL        of CS10 and label as “Blank”.    -   8.5.72 If Applicable: Recorded number of vials filled. Recorded        number of vials filled below, not including blank.    -   8.5.73 If Applicable: Environmental Monitoring. After        processing, verified BSC and personnel monitoring had been        performed

TIL Cryopreservation of Sample

-   -   8.5.74 If Applicable: Calculated Volume for Cryopreservation.        Calculated the volume of cells required to obtain 1×10⁷ cells        for cryopreservation.

Volume of Cells Total Viable TIL Viable Cell required for required forConcentration From cryopreservation cryopreservation Step 8.5.51A C = A÷ B A. 1 × 10⁷ cells B. cells/mL C. mL

-   -   8.5.75 If Applicable: Removed sample for Cryopreservation. In        the BSC, using the appropriately sized syringe, removed the        calculated volume (Step    -   8.5.74C) from the TIL Suspension transfer pack. Placed in        appropriately sized conical tube and label as “Cryopreservation        Sample 1×10⁷ cells,” dated, and lot number. Placed a red cap        (W3012845) on the TIL Suspension transfer pack.    -   8.5.76 If Applicable: Placed TIL in Incubator. Placed TIL        Suspension Transfer Pack in incubator until needed.    -   8.5.77 If Applicable: Cryopreservation sample. Centrifuged the        “Cryopreservation Sample 1×10⁷ cells” according to the following        parameters: Speed: 350×g, Time: 10:00 minutes, Temperature:        Ambient, Brake: Full (9) Acceleration: Full (9). NOTE: Ensure        proper units are set for speed and time on the centrifuge.    -   8.5.78 If Applicable: Observed conical tube. Recorded        observations: Pellet observed? Supernatant is clear? *NOTE: If        either answer is no, contact Area Management.    -   8.5.79 If Applicable: Added CS-10. In BSC, aseptically aspirate        supernatant. Gently tap bottom of tube to resuspend cells in        remaining fluid.    -   8.5.80 If Applicable: Added CS-10. Slowly added. 0.5 mL of CS10.        Recorded volume added.    -   8.5.81 If Applicable: Labeled vial. Labeled vial with QA issued        label.    -   8.5.82 If Applicable: Filled Vials. Aliquoted resuspended volume        into labeled cryovial.    -   8.5.83 If Applicable: Filled blank. Filled another vial with 0.5        mL of CS10 and label as “Blank”.    -   8.5.84 If Applicable: Recorded number of vials filled, not        including “blank”.

Cryopreservation Sample Vials Filled at −0.5 mL

-   -   8.5.85 If Applicable: Environmental Monitoring. After        processing, verify BSC and personnel monitoring have been        performed.    -   8.5.86 Review Section 8.5

8.6 Day 11—Feeder Cells

-   -   8.6.1 Obtained feeder cells. Obtained 3 bags of feeder cells        with at least two different lot numbers from LN2 freezer. Kept        cells on dry ice until ready to thaw. NOTE: Section 8.6 could be        performed concurrently with Section 8.5.    -   8.6.2 Obtained feeder cells. Recorded feeder cell information.        Confirmed that at least two different lots of feeder cells were        obtained.    -   8.6.3 Prepared waterbath or Cryotherm. Prepared water bath or        Cytotherm for Feeder Cell thaw.    -   8.6.4 Thawed Feeder Cells. Placed the Feeder Cell bags into        individual zip top bags, based on Lot number, and thawed        37.0±2.0° C. water bath or cytotherm for ˜3-5 minutes or until        ice has just disappeared. Recorded thaw times below from timer.    -   8.6.5 Feeder cell harness preparation. Welded (per Process Note        5.11) 4S-4M60 to a CC2 Cell Connect (W3012820), replacing a        single spike of the Cell Connect apparatus (B) with the 4-spike        end of the 4S-4M60 manifold at (G). Welded H to G.    -   8.6.6 If applicable: Removed media from incubator. Removed the        Feeder Cell CM2 Media transfer pack prepared in Step 8.4.34 from        the incubator.    -   8.6.7 Attached media transfer pack Weld (per Process Note 5.11)        the “Feeder Cell CM2 Media” transfer pack to a CC2 luer. NOTE:        The bag will be attached to the side of the harness with the        needless injection port.    -   8.6.8 Transfer harness. Transferred the assembly containing the        Complete CM2 Day 11 Media into the BSC.    -   8.6.9 Pool thawed feeder cells. Within the BSC, pulled 10 mL of        air into a 100 mL syringe. Used this to replace the 60 mL        syringe on the CC2.    -   8.6.10 Pool thawed feeder cells. Wiped each port on the feeder        cell bags with an alcohol pad prior to removing the cover. Spike        the three feeder bags using three of the spikes of the CC2.        NOTE: Maintained constant pressure while turning the spike in        one direction. Ensure to not puncture the side of the port.    -   8.6.11 Pool Thawed Feeder Cells. Opened the stopcock so that the        line from the feeder cell bags is open and the line to the        needless injection port is closed.    -   8.6.12 Pool Thawed Feeder Cells. Drew up the contents of the        feeder cell bags into the syringe. All three bags drained at        once. Once feeder cell bags had been drained, while maintaining        pressure on the syringe, clamped off the line to the feeder cell        bags.    -   8.6.13 Recorded volume of feeder cells. Did not detach syringe        below. the syringe from the harness. Recorded the total volume        of feeder cells in the syringe.    -   8.6.14 Added feeder cells to transfer pack. Turned the stopcock        so the line to the feeder cell bag is closed and the line to the        media Transfer Pack was open.

Ensured the line to media transfer pack is unclamped.

-   -   8.6.15 Added feeder cells to transfer pack Dispensed the feeder        cells from the syringe into the “Feeder Cell CM2 Media” transfer        pack. Clamped off the line to the transfer pack containing the        feeder cells and leave the syringe attached to the harness.    -   8.6.16 Mixed pooled feeder cells. Massaged bag to mix the pooled        feeder cells in the transfer pack.    -   8.6.17 Labeled transfer pack. Labeled bag as “Feeder Cell        Suspension” and Lot number.    -   8.6.18 Calculated total volume in Transfer Pack Calculated the        total volume of feeder cell suspension.    -   8.6.19 Removed cell count samples. Using a separate 3 mL syringe        for each sample, pulled 4×1.0 mL cell count samples from Feeder        Cell Suspension Transfer Pack using the needless injection port.        Aliquoted each sample into the cryovials labeled in Step 8.4.27.        NOTE: Wiped the needless injection port with a sterile alcohol        pad (W3009488) and mixed Feeder Cell Suspension between each        sampling for cell counts.    -   8.6.20 Performed Cell Counts. Performed cell counts and        calculations utilizing NC-200 and Process Note 5.14. Diluted        cell count samples by adding 0.5 mL of cell suspension into 4.5        mL of AIM-V media labelled with the lot number and “For Cell        Count Dilutions”. This will give a 1:10 dilution.    -   Adjusted if necessary.    -   8.6.21 Recorded Cell Count. Sample volumes    -   8.6.22 Determine Multiplication Factor

Parameter Formula Result Total cell count 8.6.21A + 8.6.21B C. μL sampleVolume Multiplication C ÷ 8.6.21A D. Factor

-   -   8.6.23 Selected protocols and entered multiplication factors.        Ensured the “Viable Cell Count Assay” protocol had been        selected, all multiplication factors, and sample and diluent        volumes had been entered.    -   8.6.24 Recorded File Name, Viability and Cell Counts from        Nucleoview.    -   8.6.25 Determined the Average of Viable Cell Concentration and        Viability of the cell counts performed.

Parameter Formula Result Viability (8.6.24A + 8.6.24B) ÷ 2 E. % ViableCell (8.6.24C + 8.6.240) ÷ 2 F. cells/mL Concentration

-   -   8.6.26 Determined Upper and Lower Limit for counts.

Parameter Formula Result Lower Limit 8.6.25F × 0.9 G. cells/mL UpperLimit 8.6.25F × 1.1 H. cells/mL

-   -   8.6.27 Were both counts within acceptable limits?

Parameter Formula Result (Yes/No) Lower Limit 8.6.24 C and D ≥ 8.6.26GUpper Limit 8.6.24 C and D ≤ 8.6.26H NOTE: If either result was “No”performed second set of counts in steps 8.6.28-8.6.35. 8.6.28 IfApplicable: Performed cell counts Perform cell counts and calculationsin utilizing NC-200 per SOP-00314 and Process Note 5.14 NOTE: Dilutioncould be adjusted according based off the expected concentration ofcells.

-   -   8.6.29 If Applicable: Recorded Cell Count, sample volumes. NOTE:        If no dilution needed, enter “Sample [μL]”=200, “Dilution        [μL]”=0.    -   8.6.30 If Applicable: Determined Multiplication Factor.

Parameter Formula Result Total cell count 8.6.29A + 8.6.29B C. mL sampleVolume Multiplication C ÷ 8.6.29A D. Factor

-   -   8.6.31 Select protocols and enter multiplication factors. Ensure        the “Viable Cell Count Assay” protocol was selected, all        multiplication factors, and sample and diluent volumes were        entered. NOTE: If no dilution needed, enter “Sample [μL]”=200,        “Dilution [μL]”=0. 8.6.32 If Applicable: Recorded Cell Counts        from Nucleoview    -   8.6.33 If Applicable: Determined the Average of Viable Cell        Concentration and Viability of the cell counts performed.

Parameter Formula Result Viability (8.6.32A + 8.6.32B) ÷ 2 E. % ViableCell (8.6.32C + 8.6.32D) ÷ 2 F. cells/mL Concentration

-   -   8.6.34 If Applicable: Determined Upper and Lower Limit for        counts.

Parameter Formula Result Lower Limit 8.6.33F × 0.9 G. cells/mL UpperLimit 8.6.33F × 1.1 H. cells/mL

-   -   8.6.35 If Applicable: Were counts within acceptable limits?

Parameter Formula Result (Yes/No) Lower Limit 8.6.32 C and D ≥ 8.6.34GUpper Limit 8.6.32 C and D ≤ 8.6.34H NOTE: If either result was “No,”continued to step 8.6.36 to find a total Average Viable CellConcentration and proceed with calculations.

-   -   8.6.36 If Applicable: Determined an average Viable Cell        Concentration from all four counts performed.    -   8.6.37 Adjusted Volume of Feeder Cell Suspension. Calculated the        adjusted volume of Feeder Cell suspension after removal of cell        count samples. Total Feeder Cell Volume from Step 8.6.18C minus        4.0 ml removed.    -   8.6.38 Calculated Total Viable Feeder Cells.

Parameter Formula Result Average Viable Cell 8.6.25 F* A. cells/mLConcentration* -or- 8.6.33 F* -or- 8.6.36 E* Total Volume 8.6.37 C B. mLTotal Viable Cells A × B C. cells

-   -   If Total Viable Cells are <5×10⁹, proceed to Step 8.6.39. If        Total Viable Cells are ≥5×10⁹, proceed to Step 8.5.70.    -   8.6.39 If Applicable: Obtained additional Feeder Cells. Obtained        an additional bag of feeder cells from LN2 freezer. Kept cells        on dry ice until ready to thaw.    -   8.6.40 If Applicable: Obtained additional Feeder Cells. Recorded        feeder cell information.    -   8.6.41 If Applicable: Thawed Additional Feeder Cells. Placed the        4th Feeder Cell bag into a zip top bag and thaw in a        37.0±2.0° C. water bath or cytotherm for ˜3-5 minutes or until        ice has just disappeared. Recorded thaw time.    -   8.6.42 If applicable: Pooled additional feeder cells. In the        BSC, pulled 10 mL of air into a new 100 mL syringe. Used this to        replace the syringe on the harness.    -   8.6.43 If applicable: Pooled additional feeder cells Wiped the        port of the feeder cell bag with an alcohol pad prior to        removing the cover. Spiked the feeder cell bag using one of the        remaining spikes of the harness prepared in Step 8.6.7 NOTE:        Maintained constant pressure while turning the spike in one        direction. Ensured to not puncture the side of the port.    -   8.6.44 If applicable: Pooled additional feeder cells. Opened the        stopcock so that the line from the feeder cell bag was open and        the line to the needless injection port was closed.    -   8.6.45 If applicable: Pooled additional feeder cells. Drew up        the contents of the feeder cell bag into the syringe. Recorded        volume.    -   8.6.46 If Applicable: Measured Volume. Measured the volume of        the feeder cells in the syringe and recorded below (B).        Calculated the new total volume of feeder cells.

Total Feeder Cell Feeder Cell Volume Feeder Cell Volume Volume from Step8.6.37C from Step 8.6.45 C = A + B A. mL B. mL C. mL

-   -   8.6.47 If Applicable: Added Feeder Cells to Transfer Pack.        Turned the stopcock so the line to the feeder cell bag was        closed and the line to the “Feeder Cell Suspension” transfer        pack was open. Ensured the line to the transfer pack was        unclamped. Dispensed the feeder cells from the syringe into the        “Feeder Cell Suspension” transfer pack. Clamped the line to the        transfer pack and left the syringe attached to the harness.    -   8.6.48 If Applicable: Added Feeder Cells to Transfer Pack.        Massaged bag to mix the pooled feeder cells in the Feeder Cell        Suspension transfer pack.    -   8.6.49 If Applicable: Prepared dilutions. In the BSC, add 4.5 mL        of AIM-V Media that has been labelled with “For Cell Count        Dilutions” and lot number to four 15 mL conical tubes. Label the        tubes with the lot number and tube number (1-4). Labeled 4        cryovials “Additional Feeder” and vial number (1-4).    -   8.6.50 If Applicable: Prepared cell counts. Using a separate 3        mL syringe for each sample, removed 4×1.0 mL cell count samples        from Feeder Cell Suspension transfer pack, using the needless        injection port. Aliquoted each sample into cryovials labeled in        Step 8.6.49. NOTE: Wiped the needless injection port with a        sterile alcohol pad and mix Feeder Cell Suspension between each        sampling for cell counts.    -   8.6.51 If Applicable: Performed Cell Counts. Performed cell        counts and calculations utilizing NC-200 and Process Note 5.14.        Diluted cell count samples by adding 0.5 mL of cell suspension        into 4.5 mL of AIM-V media labelled with the lot number and “For        Cell Count Dilutions”. This will give a 1:10 dilution. Adjusted        if necessary.    -   8.6.52 If Applicable: Recorded Cell Count sample volumes.    -   8.6.53 If Applicable: Determined Multiplication Factor

Parameter Formula Result Total cell count 8.6.52A + 8.6.52B C. μL sampleVolume Multiplication C ÷ 8.6.52A D. Factor

-   -   8.6.54 If Applicable: Selected protocols and entered        multiplication factors. Ensured the “Viable Cell Count Assay”        protocol had been selected, all multiplication factors, and        sample and diluent volumes had been entered.    -   8.6.55 If Applicable: Recorded File Name, Viability and Cell        Counts from Nucleoview.    -   8.6.56 If Applicable: Determine the Average of Viable Cell        Concentration and Viability of the cell counts performed.

Parameter Formula Result Viability (8.6.55A + 8.6.55B) ÷ 2 E. % ViableCell (8.6.55C + 8.6.55D) ÷ 2 F. cells/mL Concentration

-   -   8.6.57 If Applicable: Determine Upper and Lower Limit or counts.

Parameter Formula Result Lower Limit 8.6.56F × 0.9 G. cells/mL UpperLimit 8.6.56F × 1.1 H. cells/mL

-   -   Are both counts within acceptable limits? NOTE: If either result        is “No” perform second set of counts in Steps 8.5.59-8.5.65    -   8.6.59 If Applicable: Performed cell counts. Performed cell        counts and calculations in utilizing NC-200 and Process Note        5.14. NOTE: Dilution could be adjusted according based off the        expected concentration of cells.    -   8.6.60 If Applicable: Recorded Cell Count sample volumes. NOTE:        If no dilution was needed, entered “Sample [μL]”=200, “Dilution        [μL]”=0    -   8.6.61 If Applicable: Determined Multiplication Factor.

Parameter Formula Result Total cell count 8.6.60A + 8.6.60B C. μL sampleVolume Multiplication C ÷ 8.6.60A D. Factor

-   -   8.6.62 If Applicable: Select protocols and enter multiplication        factors. Ensured the “Viable Cell Count Assay” protocol has been        selected, all multiplication factors, and sample and diluent        volumes had been entered. NOTE: If no dilution was needed,        entered “Sample [μL]”=200, “Dilution [μL]”=0    -   8.6.63 If Applicable: Recorded Cell Counts from Nucleoview.    -   8.6.64 If Applicable: Determined the Average of Viable Cell        Concentration and Viability of the cell counts performed.

Parameter Formula Result Viability (8.6.63A + 8.6.63B) ÷ 2 E. % ViableCell (8.6.63C + 8.6.63D) ÷ 2 F. cells/mL Concentration

-   -   8.6.65 If Applicable: Determined Upper and Lower Limit for        counts

Parameter Formula Result Lower Limit 8.6.64F × 0.9 G. cells/mL UpperLimit 8.6.64F × 1.1 H. cells/mL

-   -   8.6.66 If Applicable: Were counts within acceptable limits?

Parameter Formula Result (Yes/No) Lower Limit 8.6.63 C and D ≥ 8.6.65GUpper Limit 8.6.63 C and D ≤ 8.6.65H NOTE: If either result was “No,”continue to Step 8.6.67 to find a total Average Viable CellConcentration and proceeded with calculations. 8.6.67 If Applicable:Determined an average Viable Cell Concentration from all four countsperformed.

-   -   8.6.68 If Applicable: Adjusted Volume of Feeder Cell Suspension.        Calculated the adjusted volume of Feeder Cell suspension after        removal of cell count samples. Total Feeder Cell Volume from        Step 8.6.46C minus 4.0 mL removed.    -   8.6.69 If Applicable: Calculated Total Viable Feeder Cells.

Parameter Formula Result Average Viable Cell 8.6.56 F* A. cells/mLConcentration* -or- 8.6.64 F* -or- 8.6.67 E* Total Volume 8.6.68 C B. mLTotal Viable Cells A × B C. cells *Circled step reference used todetermine Viable Cell Concentration.

-   -   8.6.70 Calculated Volume of Feeder Cells. Calculated the volume        of Feeder Cell Suspension that was required to obtain 5×10⁹        viable feeder cells.

Viable Cell Volume of Feeder Concentration from Cells = 5 × 10⁹ viableNumber of Feeder Step 8.6.38A* or cells Cells Required Step 8.6.69A* C =A ÷ B A. 5 × 10⁹ Viable Cells B. cells/mL C. mL *Circle applicable step

-   -   8.6.71 Calculated excess feeder cell volume. Calculated the        volume of excess feeder cells to remove. Round down to nearest        whole number.

Total Volume of Feeder Volume of Excess Cells in Transfer Pack Volume ofFeeder Cells = Feeder Cells to from Step 8.6.46C* 5 x 10⁹ viable cellsfrom remove. viable cells or Step 8.6.68C* Step 8.6.70C C = A − B A. mLB. mL C. mL *Circle applicable step

-   -   8.6.72 Removed excess feeder cells. In a new 100 mL syringe,        pulled up 10 mL of air and attached the syringe to the harness.    -   8.6.73 Removed excess feeder cells. Opened the line to the        “Feeder Cell Suspension” transfer pack. Using the syringe drew        up the volume of feeder cells calculated in Step 8.6.71C plus an        additional 10.0 mL from the Transfer Pack into a 100 mL syringe.        Closed the line to the Feeder Cell Suspension transfer pack once        the volume of feeder cells is removed. Did not remove final        syringe. NOTE: Once a syringe has been filled, replaced it with        a new syringe. Multiple syringes could be used to remove total        volume. With each new syringe, pulled in 10 mL of air.    -   8.6.74 Recorded volume. Recorded the total volume (including the        additional 10 mL) of feeder cells removed.    -   8.6.75 Added OKT3. In the BSC, using a 1.0 mL syringe and 16G        needle, drew up 0.15 mL of OKT3.    -   8.6.76 Added OKT3. Aseptically removed the needle from the        syringe and attach the syringe to the needless injection port.        Injected the OKT3.    -   8.6.77 Added OKT3. Opened the stopcock to the “Feeder Cell        Suspension” transfer pack and added 10 mL of feeder cells        removed in Step 8.6.73 to flush OKT3 through the line.    -   8.6.78 Added OKT3. Turned the syringe upside down and push air        through to clear the line to the Feeder Cell Suspension transfer        pack.    -   8.6.79 Added OKT3. Left the remaining feeder cell suspension in        the syringe. Closed all clamps and remove the harness from the        BSC.    -   8.6.80 Heat Sealed. Heat sealed (per Process Note 5.12) the        Feeder Cell Suspension transfer pack, leaving enough tubing to        weld. Discarded the harness.    -   8.6.81 Review Section 8.6

8.7 Day 11 G-Rex Fill and Seed

-   -   8.7.1 Set up G-Rex500MCS. Outside the BSC, removed a G-Rex500MCS        from packaging and inspected the flask for any cracks or kinks        in the tubing.

Ensured all luer connections and closures were tight. Closed all clampson the G-Rex500MCS lines except for the vent filter line. Using a markerdrew a line at the 4.5 L gradation.

-   -   8.7.2 Removed media from incubator. Removed the “Complete CM2        Day 11 Media”, prepared in Step 8.4.35, from the incubator.    -   8.7.3 Prepared to pump media. Welded (per Process Note 5.11) the        red line of the G-Rex500MCS to the repeater pump transfer set        attached to the complete CM2 Day 11 Media.    -   8.7.4 Prepare to pump media. Hung the “Complete CM2 Day 11        Media” bag on an IV pole. Fed the pump tubing through the Baxa        pump.    -   8.7.5 Pumped media into G-Rex500MCS. Set the Baxa pump to “High”        and “9”. Pumped 4.5 L of media into the G-Rex500MCS, filling to        the line marked on the flask in Step 8.7.1.    -   8.7.6 Heat sealed. Heat sealed the red line (per Process Note        5.12) of the G-Rex500MCS near the weld created in Step 8.7.3.        8.7.7 Labeled Flask. Labeled the flask with the Attach a sample        “Day 11 QA provided in-process “Day 11” label.    -   8.7.8 If applicable: Incubated flask. Held flask in incubator        while waiting to seed with TIL.    -   8.7.9 Welded the Feeder Cell: Suspension transfer pack to the        flask Sterile welded (per Process Note 5.11) the red line of the        G-Rex500MCS to the “Feeder Cell Suspension” transfer pack.    -   8.7.10 Added Feeder Cells to G-Rex500MCS. Opened all clamps        between Feeder Cell Suspension and G-Rex500MCS and added Feeder        Cell Suspension to flask by gravity feed. Ensured the line has        been completely cleared.    -   8.7.11 Heat sealed. Heat sealed (per Process Note 5.12) the red        line near the weld created in Step 8.7.9.    -   8.7.12 Welded the TIL Suspension transfer pack to the flask.        Sterile weld (per Process Note 5.11) the red line of the        G-Rex500MCS to the “TIL Suspension” transfer pack.    -   8.7.13 Added TIL to G-Rex500MCS. Opened all clamps between TIL        Suspension and G-Rex500MCS and added TIL Suspension to flask by        gravity feed. Ensured the line has been completely cleared.    -   8.7.14 Heat sealed. Heat sealed (per process note 5.12) the red        line near the weld created in Step 8.7.12 to remove the TIL        suspension bag.    -   8.7.15 Incubated G-Rex500MCS. Checked that all clamps on the        G-Rex500MCS were closed except the large filter line and place        in the incubator. Incubator parameters: Temperature LED Display:        37.0±2.0° C., CO2 Percentage: 5.0±1.5% CO2.    -   8.7.16 Calculated incubation window. Performed calculations to        determine the proper time to remove G-Rex500MCS from incubator        on Day 16. Time of incubation (Step 8.7.15). Lower limit: Time        of incubation+108 hours.    -   Upper limit: Time of incubation+132 hours.    -   8.7.17 Environmental Monitoring. After processing, verified BSC        and personnel monitoring had been performed.    -   8.7.18 Submit Samples. Submit samples to Login.    -   8.7.19 Review Section 8.7

8.8 Day 11 Excess TIL Cryopreservation

-   -   8.8.1 If Applicable: Froze Excess TIL Vials. Verified the CRF        has been set up prior to freeze. Perform Cryopreservation.    -   8.8.2 If Applicable: Started CRF. Recorded the total number of        vials placed into the CRF (not including blank). Verify number        of vials transferred into the CRF matches total number of vials        prepared in Step 8.5.72 or Step 8.5.84 Step 8.5.72C or Step        8.5.84    -   8.8.3 If applicable: Initiated automated portion of the freezing        profile. Recorded START TIME for the initiation of the automated        portion of the freezing profile.    -   8.8.4 If Applicable: Transferred vials from Controlled Rate        Freezer to the appropriate storage. Upon completion of freeze,        transfer vials from CRF to the appropriate storage container.    -   8.8.5 If applicable: Transferred vials to appropriate storage.        Recorded storage location in LN2.    -   8.8.6 Review Section 8.8

8.9 Day 16 Media Preparation

-   -   8.9.1 Pre-warmed AIM-V Media. Removed three CTS AIM V 10 L media        bags from 2-8° C. at least 12 hours prior to use and place at        room temperature protected from light. Verify each bag is within        expiry. Labeled each bag with Bag Number (1-3), lot number,        date, and “warming start time HHMM”. Record warming start time        and date.    -   8.9.2 Calculated time Media from step 8.9.1 was warmed.        Calculated the warming time of media bags 1, 2, and 3 from step        8.9.1. Ensured all bags have been warmed for a duration between        12 and 24 hours.    -   8.9.3 Checked room sanitization, line clearance, and materials.        Confirmed room sanitization, line clearance, and materials.    -   8.9.4 Ensured completion of pre-processing table.    -   8.9.5 Environmental Monitoring. Prior to processing, ensured        pre-process environmental monitoring had been initiated.    -   8.9.6 Setup 10 L Labtainer for Supernatant. In the BSC attached        the larger diameter end of a fluid pump transfer set to one of        the female ports of a 10 L Labtainer bag using the Luer        connectors.    -   8.9.7 Setup 10 L Labtainer for Supernatant Label as        “Supernatant” and Lot number.    -   8.9.8 Setup 10 L Labtainer for Supernatant Ensure all clamps        were closed prior to removing from the BSC. NOTE: Supernatant        bag was used during TIL Harvest (Section 8.10), which may be        performed concurrently with media preparation.    -   8.9.9 Thawed IL-2. Thawed 5×1.1 mL aliquots of IL-2 (6×10⁶        IU/mL) (BR71424) per bag of CTS AIM V media until all ice had        melted.    -   Recorded IL-2 Lot number and Expiry. Attached IL-2 labels.    -   8.9.10 Aliquoted GlutaMax. In BSC, aliquoted 100.0 mL of        Glutamax into an appropriately sized receiver. Recorded the        volume added to each receiver NOTE: Initially prepared one bag        of AIM-V media following Step 8.9.10—Step 8.9.28. Additional        bags required were determined in Step 8.10.59.    -   8.9.11 Labeled receivers. Labeled each receiver as “GlutaMax.”    -   8.9.12 Added IL-2 to GlutaMax. Using a micropipette, added 5.0        mL of IL-2 to each GlutaMax receiver. Ensured to rinse the tip        per process note 5.18 and used a new pipette tip for each mL        added. Recorded volume added to each Glutamax receiver below.    -   8.9.13 Labeled receivers. Labeled each receiver as        “GlutaMax+IL-2” and receiver number.    -   8.9.14 Prepared CTS AIM V media bag for formulation. Ensured CTS        AIM V 10 L media bag (W3012717) was warmed at room temperature        and protected from light for 12-24 hours prior to use. Recorded        end incubation time in Step 8.9.2.    -   8.9.15 Prepared CTS AIM V media bag for formulation. In the BSC,        closed clamp on a 4″ plasma transfer set, then connected to the        bag using the spike ports. NOTE: Maintained constant pressure        while turning the spike in one direction. Ensured to not        puncture the side of the port.    -   8.9.16 Prepared CTS AIM V media bag for formulation. Connected        the larger diameter end of a repeater pump fluid transfer set to        the 4″ plasma transfer set via luer.    -   8.9.17 Stage Baxa Pump. Stage Baxa pump next to BSC. Removed        pump tubing section of repeater pump fluid transfer set from BSC        and installed in repeater pump.    -   8.9.18 Prepared to formulate media. In BSC, removed syringe from        Pumpmatic Liquid-Dispensing System (PLDS) and discarded. NOTE:        Ensured to not compromise the sterility of the PLDS pipette.    -   8.9.19 Prepared to formulate media. Connected PLDS pipette to        smaller diameter end of repeater pump fluid transfer set via        luer connection and placed pipette tip in “GlutaMax+IL-2”        prepared in Step 8.9.13 for aspiration Open all clamps between        receiver and 10 L bag.    -   8.9.20 Pumped GlutaMax+IL-2 into bag. Set the pump speed to        “Medium” and “3” and pump all “GlutaMax+IL-2” into 10 L CTS AIM        V media bag. Once no solution remains, clear line and stop pump.        Recorded the volume of GlutaMax containing IL-2 added to each        Aim V bag below.Recorded/    -   8.9.21 Removed PLDS. Ensured all clamps were closed, and removed        the PLDS pipette from the repeater pump fluid transfer set.        Removed repeater pump fluid transfer set and red cap the 4″        plasma transfer set.    -   8.9.22 Labeled Bags. Labeled each bag of “Complete CM4 Day 16        media” prepared.    -   8.9.23 Removed Media Retain per Sample Plan. Using a 30 mL        syringe, removed 20.0 mL of “Complete CM4 Day 16 media” by        attaching syringe to the 4″ plasma transfer set and dispensed        sample into a 50 mL conical tube.    -   NOTE: Only removed the Media Retain Sample from the first bag of        media prepared. NOTE: Ensure 4″ plasma transfer set was either        clamped or red capped after removal of syringe.    -   8.9.24 Attached new repeater pump fluid transfer set. Attached        the larger diameter end of a new fluid pump transfer set onto        the 4″ plasma transfer set that was connected to the “Complete        CM4 Day 16 media” bag.    -   8.9.25 Labeled and stored sample. Labeled with sample plan        inventory label and stored media retain sample at 2-8° C. until        submitted to Login for testing per Sample Plan.    -   8.9.26 Signed for Sampling. Ensured that LIMS sample plan sheet        was completed for removal of the sample.    -   8.9.27 Monitor Incubator. Monitored Incubator. If applicable,        per Step    -   8.9.10, monitor for additional bags prepared. Incubator        parameters: Temperature LED Display: 37.0±2.0° C., CO2        Percentage: 5.0±1.5% CO2.    -   8.9.28 Warmed Complete CM4 Day 16 Media. Warmed the first bag of        Complete CM4 Day 16 Media in incubator for ≥30 minutes until        ready for use. If applicable, per Step 8.10.59, warmed        additional bags.    -   8.9.29 Prepared Dilutions. In the BSC, added 4.5 mL of AIM-V        Media that had been labelled with Batch record Lot Number and        “For Cell Count Dilutions” to each 4×15 mL conical tube. Labeled        the conical tubes with the lot number and tube number (1-4).        Labeled 4 cryovials with vial number (1-4). Kept vials under BSC        to be used in Step 8.10.31.    -   8.9.30 Reviewed Section 8.9

8.10 Day 16 REP Spilt

-   -   8.10.1 Pre-processing table.    -   8.10.2 Monitored Incubator. Monitored Incubator. Incubator        parameters: Temperature LED Display: 37.0±2.0° C., CO2        Percentage: 5.0±1.5% CO2    -   8.10.3 Removed G-Rex500MCS from Incubator. Performed check below        to ensure incubation parameters are met before removing        G-Rex500MCS from incubator.

Time of Lower limit Upper limit Removal Is 8.7.16B < Time from from fromof Removal from Step 8.7.16B Step 8.7.16C incubator incubator < Step(DDMMMYY (DDMMMYY (DDMMMYY 8.7.16C HHMM) HHMM) HHMM) Yes/No*

-   -   Removed G-Rex500MCS from the incubator.    -   8.10.4 Setup 1 L Transfer Pack. Heat sealed a 1 L transfer pack        (W3006645) per Processed Note 5.12, leaving ˜12″ of line.    -   8.10.5 Prepared 1 L Transfer Pack. Labeled 1 L transfer pack as        TIL Suspension.    -   8.10.6 Weighed 1 L Transfer Pack Place 1 L transfer pack,        including the entire line, on a scale and record dry weight.    -   8.10.7 GatheRex Setup. Sterile welded (per Process Note 5.11)        the red media removal line from the G-Rex500MCS to the repeater        pump transfer set on the 10 L labtainer bag “Supernatant”        prepared in Step 8.9.8. Sterile welded the clear cell removal        line from the G-Rex500MCS to the TIL Suspension transfer pack        prepared in Step 8.10.5.    -   8.10.8 GatheRex Setup. Placed G-Rex500MCS flask on the left side        of the GatheRex. Placed the supernatant labtainer bag and TIL        suspension transfer pack to the right side.    -   8.10.9 GatheRex Setup. Installed the red media removal line from        the G-Rex500MCS to the top clamp (marked with a red line) and        tubing guides on the GatheRex. Installed the clear harvest line        from the G-Rex500MCS to the bottom clamp (marked with a blue        line) and tubing guides on the GatheRex.    -   8.10.10 GatheRex Setup. Attached the gas line from the GatheRex        to the sterile filter of the G-Rex500 MCS. NOTE: Before removing        the supernatant from the G-Rex500MCS, ensured all clamps on the        cell removal lines were closed.    -   8.10.11 Volume Reduction of G-Rex500MCS. Transferred −4.5 L of        culture supernatant from the G-Rex500MCS to the 10 L Labtainer        per SOP-01777. Visually inspect G-Rex500MCS to ensure flask as        level and media had been reduced to the end of the aspirating        dip tube. NOTE: If the GatheRex stops prematurely, it could be        restarted by pressing the button with the arrow pointing to the        right again.    -   8.10.12 Prepared flask for TIL Harvest. After removal of the        supernatant, closed all clamps to the red line.    -   8.10.13 Initiation of TIL Harvest. Recorded the start time of        the TIL harvest.    -   8.10.14 Initiation of TIL Harvest. Vigorously tap flask and        swirl media to release cells. Performed an inspection of the        flask to ensure all cells have detached. NOTE: Contact area        management if cells did not detach.    -   8.10.15 Initiation of TIL Harvest. Tilted the flask to ensure        hose is at the edge of the flask. Note: If the cell collection        straw is not at the junction of the wall and bottom membrane,        rapping the flask while tilted at a 450 angle is usually        sufficient to properly position the straw.    -   8.10.16 TIL Harvest. Released all clamps leading to the TIL        suspension transfer pack.    -   8.10.17 TIL Harvest. Using the GatheRex transferred the cell        suspension into the TIL Suspension transfer pack. NOTE: Be sure        to maintain the tilted edge until all cells and media are        collected.    -   8.10.18 TIL Harvest. Inspected membrane for adherent cells.    -   8.10.19 Rinse flask membrane. Rinse the bottom of the        G-Rex500MCS. Cover −1/4 of gas exchange membrane with rinse        media.    -   8.10.20 Closed clamps on G-Rex500MCS. Ensured all clamps are        closed on the G-Rex500MCS.    -   8.10.21 Heat sealed. Heat sealed (per Process Note 5.12) the        Transfer Pack containing the TIL as close to the weld as        possible so that the overall tubing length remained        approximately the same.    -   8.10.22 Heat sealed. Heat sealed the 10 L Labtainer containing        the supernatant (per Process Note 5.12) and passed into the BSC        for sample collection in Step 8.10.25.    -   8.10.23 Calculated volume of TIL suspension. Recorded weight of        Transfer Pack with cell suspension and calculate the volume        suspension.    -   8.10.24 Prepared transfer pack for sample removal. Welded (per        Process Note 5.11) a 4″ Plasma Transfer Set, to the TIL        Suspension transfer pack from Step 8.10.21, leaving the female        luer end attached as close to the bag as possible.    -   8.10.25 Removed testing samples from cell supernatant. In the        BSC, remove 10.0 mL of supernatant from 10 L labtainer using        female luer port and appropriately sized syringe. Placed into a        15 mL conical tube and label as “BacT” Retain the tube for BacT        sample in Step 8.10.28.    -   8.10.26 Removed testing samples from cell supernatant. Using a        separate syringe, removed 10.0 mL of supernatant and placed into        a 15 mL conical tube. Retained the tube for mycoplasma sample        for use in Step    -   8.10.32. Labeled tube as “Mycoplasma diluent”    -   8.10.27 Closed supernatant bag. Placed a red cap on the luer        port to close the bag, and pass out of BSC.    -   8.10.28 Sterility & BacT Testing Sampling. In the BSC, removed a        1.0 mL sample from the 15 mL conical labeled BacT prepared in        Step 8.10.25 using an appropriately sized syringe and inoculate        the anaerobic bottle. Repeat the above for the aerobic bottle.        NOTE: This step may be performed out of sequence.    -   8.10.29 Labeled and store samples. Labeled with sample plan        inventory label and store BacT sample at room temperature,        protected from light, until submitted to Login for testing per        Sample Plan. NOTE: Did not cover barcode on bottle with label.    -   8.10.30 Signed for Sampling. Ensured that LIMS sample plan sheet        is completed for removal of the sample.    -   8.10.31 Removed Cell Count Samples. In the BSC, using separate 3        mL syringes for each sample, removed 4×1.0 mL cell count samples        from “TIL Suspension” transfer pack using the luer connection.        Placed samples in cryovials prepared in Step 8.9.29.    -   8.10.32 Removed Mycoplasma Samples. Using a 3 mL syringe,        removed 1.0 mL from TIL Suspension transfer pack and place into        15 mL conical labeled “Mycoplasma diluent” prepared in Step        8.10.26.    -   8.10.33 Label and store sample. Labeled with sample plan        inventory label and stored Mycoplasma sample at 2-8° C. until        submitted to Login for testing per Sample Plan.    -   8.10.34 Signed for Sampling. Ensured that LIMS sample plan sheet        was completed for removal of the sample.    -   8.10.35 Prepared Transfer Pack for Seeding. In the BSC, attached        the large diameter tubing end of a Repeater Pump Fluid Transfer        Set to the Luer adapter on the transfer pack containing the TIL.        Clamped the line close to the transfer pack using a hemostat.        Placed a red cap onto the end of the transfer set.    -   8.10.36 Placed TIL in Incubator. Removed cell suspension from        the BSC and place in incubator until needed. Recorded time.    -   8.10.37 Performed Cell Counts. Performed cell counts and        calculations utilizing NC-200 and Process Note 5.14. Diluted        cell count samples initially by adding 0.5 mL of cell suspension        into 4.5 mL of AIM-V media prepared in Step 8.9.29. This gave a        1:10 dilution.    -   8.10.38 Recorded Cell Count sample volumes    -   8.10.39 Determined Multiplication Factor.

Parameter Formula Result Total cell count 8.10.38A + 8.10.38B C. μLsample Volume Multiplication C ÷ 8.10.38A D. Factor

-   -   8.10.40 Selected protocols and enter multiplication factors.        Ensured the “Viable Cell Count Assay” protocol had been        selected, all multiplication factors, and sample and diluent        volumes had been entered.    -   8.10.41 Recorded File Name, Viability and Cell Counts from        Nucleoview.    -   8.10.42 Determined the Average of Viable Cell Concentration and        Viability of the cell counts performed.

Parameter Formula Result Viability (8.10.41A + 8.10.41B) ÷ 2 E. % ViableCell (8.10.41C + 8.10.41D) ÷ 2 F. cells/mL Concentration

-   -   8.10.43 Determined Upper and Lower Limit for counts.

Parameter Formula Result Lower Limit 8.10.42F × 0.9 G. cells/mL UpperLimit 8.10.42F × 1.1 H. cells/mL

-   -   8.10.44 Were both counts within acceptable limits?

Parameter Formula Result (Yes/No) Lower Limit 8.10.41C and D ≥ 8.10.43GUpper Limit 8.10.41C and D ≤ 8.10.43H

-   -   8.10.45 If Applicable: Performed cell counts. Performed cell        counts and calculations in utilizing NC-200 and Process Note        5.14. NOTE: Dilution may be adjusted according based off the        expected concentration of cells.    -   8.10.46 If Applicable: Recorded Cell Count sample volumes. NOTE:        If no dilution was needed, enter “Sample [μL]”=200, “Dilution        [μL]”=0    -   8.10.47 If Applicable: Determined Multiplication Factor.

Parameter Formula Result Total cell count 8.10.46A + 8.10.46B C. mLsample Volume Multiplication C ÷ 8.10.46A D. Factor

-   -   8.10.48 If Applicable: Select protocols and enter multiplication        factors.        -   Ensure the “Viable Cell Count Assay” protocol has been            selected, all multiplication factors, and sample and diluent            volumes have been entered.    -   NOTE: If no dilution needed, enter “Sample [μL]”=200, “Dilution        [μL]”=0    -   8.10.49 If Applicable: Recorded Cell Counts from Nucleoview    -   8.10.50 If Applicable: Determined the Average of Viable Cell        Concentration and Viability of the cell counts performed.

Parameter Formula Result Viability (8.10.49A + 8.10.49B) ÷ 2 E. % ViableCell (8.10.49C + 8.10.49D) ÷ 2 F. cells/mL Concentration

-   -   8.10.51 If Applicable: Determined Upper and Lower Limit for        counts.

Parameter Formula Result Lower Limit 8.10.50F × 0.9 G. cells/mL UpperLimit 8.10.50F × 1.1 H. cells/mL

-   -   8.10.52 If Applicable: Were counts within acceptable limits?

Parameter Formula Result (Yes/No) Lower Limit 8.10.49 C and D ≥ 8.10.51GUpper Limit 8.10.49 C and D ≤ 8.10.51H NOTE: If either result is “No”continue to Step 8.10.53 to determine an average of all cell countscollected.

-   -   8.10.53 If Applicable: Determined an average Viable Cell        Concentration from all four counts performed.    -   8.10.54 Adjusted Volume of TIL Suspension. Calculated the        adjusted volume of TIL suspension after removal of cell count        samples. Total TIL Cell Volume from Step 8.10.23C minus 5.0 mL        removed for testing.    -   8.10.55 Calculated Total Viable TIL Cells.

Parameter Formula Result Average Viable Cell 8.10.42 F* A. cells/mLConcentration* -or- 8.10.50 F* -or- 8.10.53E* Total Volume 8.10.54 C B.mL Total Viable Cells A × B C. cells

-   -   8.10.56 Calculated flasks for subculture. Calculated the total        number of flasks to seed. NOTE: Rounded the number of        G-Rex500MCS flasks to see up to the neared whole number.

Number Total Viable Cell Count Target of G-Rex500MCS from Step 8.10.55CCells Required per Flask Flasks to Seed A B C = A ÷ B cells 1.0 × 10⁹cells/flask flasks NOTE: The maximum number of G-Rex500MCS flasks toseed was five. If the calculated number of flasks to seed exceeded five,only five were seeded USING THE ENTIRE VOLUME OF CELL SUSPENSIONAVAILABLE

-   -   8.10.57 Calculate number of flasks for subculture

Criteria Yes/No Number of G-Rex500MCS Flasks to Seed Step 8.10.56C 5 ≤ 5If yes, seed number of flasks calculated in Step 8.10.58. Number ofG-Rex500MCS Flasks to Seed Step 8.10.56C > 5 If yes, seed 5 flasks withALL available cells.

-   -   8.10.58 QA Review of Cell Count calculations performed in steps        8.10.38-8.10.57.    -   8.10.59 Determined number of additional media bags needed.    -   Calculated the number of media bags required in addition to the        bag prepared in Step 8.9.28.

Number of G-Rex500MCS Number of Number of Bags Number of Flasks to SeedMedia Bag Prepared in Additional Bags (Step 8.10.56C) Required Step8.9.22 to Prepare A B = A ÷ 2* C D = B − C 1 *Round the number of mediabags required up to the next whole number.

-   -   8.10.60 If Applicable: Prepared additional media. Prepared one        10 L bag of “CM4 Day 16 Media” for every two G-Rex-500M flask        needed calculated in Step 8.10.59D. Proceeded to Step 8.10.62        and seeded the first GREX-500M flask(s) while additional media        is prepared and warmed.    -   8.10.61 If Applicable: Prepared additional media bags. Prepared        and warmed the calculated number of additional media bags        determined in Step 8.10.59D, repeating Step 8.9.10—Step 8.9.28.    -   8.10.62 Filled G-Rex500MCS. Opened a G-Rex500MCS on the benchtop        and inspected for cracks in the vessel or kinks in the tubing.        Ensured all luer connections and closures were tight. Made a        mark at the 4500 mL line on the outside of the flask with a        marker. Closed all clamps on the G-Rex500MCS except the large        filter line.    -   8.10.63 Filled G-Rex500MCS. Sterile welded (per Process Note        5.11) the red media line of a G-Rex500MCS to the fluid transfer        set on the media bag prepared in Step 8.9.28.    -   8.10.64 Prepared to pump media. Hung “CM4 Day 16 Media” on an IV        pole. Fed the pump tubing through the Baxa pump.    -   8.10.65 Pump media into G-Rex500MCS. Set the Baxa pump on “High”        and “9” and pump 4500 mL of media into the flask. Pumped 4.5 L        of “CM4 Day 16 Media” into the G-Rex500MCS, filling to the line        marked on the flask in Step 8.10.62. Once 4.5 L of media had        been transferred, stopped the pump.    -   8.10.66 Heat Sealed. Heat sealed (per Process Note 5.12) the red        media line of G-Rex500MCS, near the weld created in Step        8.10.63, removing the media bag.    -   8.10.67 Repeated Fill. Repeat Steps 8.10.62-8.10.66 for each        flask calculated in Step 8.10.56C as media is warmed and        prepared for use.    -   NOTE: Multiple flasks may be filled at the same time using        gravity fill or multiple pumps. NOTE: Fill only two flasks per        bag of media.    -   8.10.68 Recorded and labelled flask(s) filled. Labeled each        flask alphabetically as it is filled and with QA provided        in-process “Day 16” labels.    -   8.10.69 Sample Labeled. Attached a sample “Day 16” label below.    -   8.10.70 If applicable: Incubated flask. Held flask in incubator        while waiting to seed with TIL.    -   8.10.71 Verified Number of Flasks Filled. Recorded the total        number of flasks filled.    -   8.10.72 Calculated volume of cell suspension to add. Calculated        the target volume of TIL suspension to add to the new        G-Rex500MCS flasks.

Total Volume of TIL Target Volume of cell suspension from Step Number offlask(s) suspension to transfer 8.10.54C filled to each flask A fromStep 8.10.71 C = A ÷ B mL mL

-   -   8.10.56C exceeds five only five will be seeded, USING THE ENTIRE        VOLUME OF CELL SUSPENSION.    -   8.10.73 Prepared Flasks for Seeding. Removed G-Rex500MCS from        Step 8.10.70 from the incubator.    -   8.10.74 Prepared for pumping. Closed all clamps on G-Rex500MCS        except large filter line. Fed the pump tubing through the Baxa        pump.    -   8.10.75 Removed TIL from incubator. Removed “TIL Suspension”        transfer pack from the incubator and record incubation end time        in Step 8.10.36.    -   8.10.76 Prepared cell suspension for seeding. Sterile welded        (per Process Note 5.11) “TIL Suspension” transfer pack from Step        8.10.75 to pump inlet line.    -   8.10.77 Tared scale. Placed TIL suspension bag on a scale.        Primed the line from the TIL suspension bag to the weld using        the Baxa pump set to “Low” and “2”. Tared the scale.    -   8.10.78 Seeded flask with TIL Suspension. Set Baxa pump to        “Medium” and “5”. Pump the volume of TIL suspension calculated        in Step    -   8.10.72C into flask. Record the volume of TIL Suspension added        to each flask.    -   8.10.79 Heat sealed. Heat sealed (per Process Note 5.12) the        “TIL Suspension” transfer pack, leaving enough tubing to weld on        the next flask. Used the line stripper to clear the residual TIL        suspension in the G-Rex flask line into the vessel.    -   8.10.80 Filled remaining flasks. Between each flask seeded,        ensured to mix “TIL Suspension” transfer pack and repeat Steps        8.10.76-8.10.79 to seed all remaining flaks. Filled flask(s) in        alphabetical order.    -   8.10.81 Monitored Incubator. NOTE: If flasks must be split among        two incubators, ensure to monitor both. Incubator parameters:        Temperature LED Display: 37.0±2.0° C., CO2 Percentage: 5.0±1.5%        CO2.    -   8.10.82 Incubated Flasks. Recorded the time each flask is placed        in the incubator.    -   8.10.83 Calculated incubation window. Performed calculations        below to determine the time range to remove G-Rex500MCS from        incubator on Day 22.

8.10.83B 8.10.83C 8.10.83A Lower limit: Upper limit: Time of Time ofTime of incubation incubation + incubation + (Step 8.10.82) 132 hours156 hours (DDMMMYY (DDMMMYY (DDMMMYY Flask HHMM) HHMM) HHMM)

-   -   8.10.84 Environmental Monitoring. After processing, verified BSC        and personnel monitoring had been performed.    -   8.10.85 Sample Submission. Ensured all Day 16 Samples were        submitted to Login.    -   8.10.86 Reviewed Section 8.10.

8.11 Day 22 Wash Buffer Preparation

-   -   8.11.1 Checked room sanitization, line clearance, and materials.    -   8.11.2 Ensured completion of pre-processing checklist.    -   8.11.3 Environmental monitoring. Prior to processing, ensured        pre-process environmental monitoring had been performed.    -   8.11.4 Prepared 10 L Labtainer Bag In BSC, attach a 4″ plasma        transfer set to a 10 L Labtainer Bag via luer connection.    -   8.11.5 Prepared 10 L Labtainer Bag Label as “Supernatant”, lot        number, and initial/date.    -   8.11.6 Prepared 10 L Labtainer Bag. Closed all clamps before        transferring out of the BSC. NOTE: Prepared one 10 L Labtainer        Bag for every two G-Rex500MCS flasks to be harvested. NOTE:        Supernatant bag(s) were used in Section 8.12, which could be run        concurrently with Section 8.11.    -   8.11.7 Welded fluid transfer set. Outside the BSC, closed all        clamps on 4S-4M60. Welded (per Process Note 5.11) repeater fluid        transfer set to one of the male luer ends of 4S-4M60.    -   8.11.8 Passed materials into the BSC. Passed Plasmalyte-A and        Human Albumin 25% into the BSC. Pass the 4S-4M60 and repeater        fluid transfer set assembly into the BSC.

Component Description Amount Needed Plasmalyte-A 3000.0 mL Human Albumin25%  120.0 mL 4S-4M60 with Repeater    1 Apparatus Fluid Transfer SetStep 8.11.7

-   -   8.11.9 Pumped Plasmalyte into 3000 mL bag. Spiked three bags of        Plasmalyte-A to the 4S-4M60 Connector set. NOTE: Wipe the port        cover with an alcohol swab (W3009488) prior to removing. NOTE:        Maintain constant pressure while turning the spike in one        direction. Ensure to not puncture the side of the port.    -   8.11.10 Pumped Plasmalyte into 3000 mL bag. Connected an Origen        3000 mL collection bag via luer connection to the larger        diameter end of the repeater pump transfer set.    -   8.11.11 Pumped Plasmalyte into 3000 mL bag. Closed clamps on the        unused lines of the 3000 mL Origen Bag.    -   8.11.12 Pumped Plasmalyte into 3000 mL bag. Staged the Baxa pump        next to the BSC. Fed the transfer set tubing through the Baxa        pump situated outside of the BSC. Set pump to “High” and “9”.    -   8.11.13 Pumped Plasmalyte into 3000 mL bag. Opened all clamps        from the Plasmalyte-A to the 3000 mL Origen Bag.    -   8.11.14 Pump Plasmalyte into 3000 mL bag. Pumped all of the        Plasmalyte-A into the 3000 mL Origen bag. Once all the        Plasmalyte-A had been transferred, stopped the pump.    -   8.11.15 Pumped Plasmalyte into 3000 mL bag. If necessary,        removed air from 3000 mL Origen bag by reversing the pump and        manipulating the position of the bag.    -   8.11.16 Pumped Plasmalyte into 3000 mL bag. Closed all clamps.        Remove the 3000 mL bag from the repeater pump fluid transfer set        via luer connection and placed a red cap (W3012845) on the line        to the bag.    -   8.11.17 Added Human Albumin 25% to 3000 mL Bag. Opened vented        mini spike. Without compromising sterility of spike, ensured        blue cap is securely fastened.    -   8.11.18 Added Human Albumin 25% to 3000 mL Bag. Spiked the        septum of a Human Albumin 25% bottle with the vented mini spike.        NOTE: Ensured to not compromise the sterility of the spike.    -   8.11.19 Added Human Albumin 25% to 3000 mL Bag. Repeated Step        8.11.17—Step 8.11.18 two times for a total of three (3) spiked        Human Albumin 25% bottles.    -   8.11.20 Added Human Albumin 25% to 3000 mL Bag. Removed the blue        cap from one vented mini spike and attach a 60 mL syringe to the        Human Serum Albumin 25% bottle.    -   8.11.21 Added Human Albumin 25% to 3000 mL Bag. Draw up 60 mL of        Human Serum Albumin 25%. NOTE: It may be necessary to use more        than one bottle of Human Serum Albumin 25%. If necessary,        disconnect the syringe from the vented mini spike and connect it        to the next vented mini spike in a Human Serum Albumin 25%        bottle. Do not remove vented mini spike from the Human Serum        Albumin 25% bottle.    -   8.11.22 Added Human Albumin 25% to 3000 mL Bag. Once 60 mL has        been obtained, remove the syringe from the vented mini spike.    -   8.11.23 Added Human Albumin 25% to 3000 mL Bag. Attach syringe        to needleless injection port on 3000 mL Origen bag filled with        Plasmalyte-A in Step 8.11.16. Dispensed all of the Human Albumin        25%. NOTE: Wiped needless injection port with an alcohol pad        before each use.    -   8.11.24 Added Human Albumin 25% to 3000 mL Bag. Repeated Step        8.11.20—Step 8.11.23 to obtain a final volume of 120.0 mL of        Human Albumin 25%.    -   8.11.25 Mixed Bag. Gently mixed the bag after all of the Human        Albumin 25% had been added.    -   8.11.26 Labeled Bag. Labeled as “LOVOWash Buffer” and lot        number, and assign a 24 hour expiry.    -   8.11.27 Prepared IL-2 Diluent. Using a 10 mL syringe, removed        5.0 mL of LOVO Wash Buffer using the needleless injection port        on the LOVO Wash Buffer bag. Dispensed LOVO wash buffer into a        50 mL conical tube and label as “IL-2 Diluent” and the lot        number. NOTE: Wiped the needless injection port with an alcohol        pad before each use.    -   8.11.28 CRF Blank Bag LOVO Wash Buffer Aliquotted. Using a 100        mL syringe, drew up 70.0 mL of LOVO Wash Buffer from the        needleless injection port. NOTE: Wiped the needless injection        port with an alcohol pad before each use.    -   8.11.29 CRF Blank Bag LOVO Wash Buffer Aliquotted. Placed a red        cap on the syringe and label as “blank cryo bag” and lot number.        NOTE: Held the syringe at room temp until needed in Step 8.14.3    -   8.11.30 Completed Wash Buffer Prep. Closed all clamps on the        LOVO Wash Buffer bag.    -   8.11.31 Thawed IL-2. Thawed one 1.1 mL of IL-2 (6×10⁶ IU/mL) ),        until all ice has melted. Record IL-2 Lot number and Expiry.        NOTE: Ensured IL-2 label is attached.    -   8.11.32 IL-2 Preparation. Added 50 μL IL-2 stock (6×10⁶ IU/mL)        to the 50 mL conical tube labeled “IL-2 Diluent.”    -   8.11.33 IL-2 Preparation. Relabeled conical as “IL-2 6×10⁴”, the        date, lot number, and 24 hour expiry. Cap and store at 2-8° C.    -   8.11.34 Cryopreservation Prep. Placed 5 cryo-cassettes at        2-8° C. to precondition them for final product cryopreservation.    -   8.11.35 Prepared Cell Count Dilutions. In the BSC, added 4.5 mL        of AIM-V Media that has been labelled with lot number and “For        Cell Count Dilutions” to 4 separate 15 mL conical tubes. Labeled        the tubes with the batch record lot number and tube number        (1-4). Set aside for use in Step 8.12.34    -   8.11.36 Prepared Cell Counts. Labeled 4 cryovials with vial        number (1-4). Kept vials under BSC to be used in Step 8.12.33.        8.11.37 Reviewed Section 8.11

8.12 Day 22 TIL Harvest

-   -   8.12.1 Monitor Incubator. Monitored the incubator. Incubator        Parameters Temperature LED display: 37±2.0° C., CO2 Percentage:        5%±1.5%. NOTE: Section 8.12 could be run concurrently with        Section 8.11.    -   8.12.2 Removed G-Rex500MCS Flasks from Incubator. Performed        check below to ensure incubation parameters were met before        removing G-Rex500MCS from incubator.

Is 8.10.83 B < Lower limit Upper limit Time of Time of from Step fromStep Removal from Removal from 8.10.83 B 8.10.83 C Incubator Incubator <(DDMMMYY (DDMMMYY (DDMMMYY Step 8.10.83 C Flask Shelf HHMM) HHMM) HHMM)Yes/No* NOTE: This step must was performed as each flask is removed fromthe incubator.

-   -   8.12.3 Prepared TIL collection bag Labeled a 3000 mL collection        bag as “TIL Suspension”, lot number, and initial/date.    -   8.12.4 Sealed off extra connections. Heat sealed off two leur        connections on the collection bag near the end of each        connection per Process Note 5.12.    -   8.12.5 GatheRex Setup. Sterile welded (per Process Note 5.11)        the red media removal line from the G-Rex500MCS to the 10 L        labtainer bag prepared in Step 8.11.5. NOTE: Referenced Process        Note 5.16 for use of multiple GatheRex devices.    -   8.12.6 GatheRex Setup. Sterile welded (per Process Note 5.11)        the clear cell removal line from the G-Rex500MCS to the TIL        Suspension collection bag prepared in Step 8.12.3. NOTE:        Reference Process Note 5.16 for use of multiple GatheRex        devices.    -   8.12.7 GatheRex Setup. Placed the G-Rex500MCS flask on the left        side of the GatheRex. Placed the supernatant Labtainer bag and        pooled TIL suspension collection bag to the right side.    -   8.12.8 GatheRex Setup. Installed the red media removal line from        the G-Rex500MCS to the top clamp (marked with a red line) and        tubing guides on the GatheRex. Installed the clear harvest line        from the G-Rex500MCS to the bottom clamp (marked with a blue        line) and tubing guides on the GatheRex.    -   8.12.9 GatheRex Setup. Attached the gas line from the GatheRex        to the sterile filter of the G-Rex500MCS. NOTE: Before removing        the supernatant from the G-Rex500MCS, ensured all clamps on the        cell removal lines were closed.    -   8.12.10 Volume Reduction. Transferred −4.5 L of supernatant from        the G-Rex500MCS to the Supernatant bag. Visually inspected        G-Rex500MCS to ensure flask is level and media had been reduced        to the end of the aspirating dip tube. Repeat step if needed.        NOTE: If the GatheRex stopped prematurely, it may be restarted        by pressing the button with the arrow pointing to the right        again.    -   8.12.11 Prepared flask for TIL Harvest. After removal of the        supernatant, closed all clamps to the red line.    -   8.12.12 Initiated collection of TIL. Recorded the start time of        the TIL harvest.    -   8.12.13 Initiated collection of TIL. Vigorously tap flask and        swirl media to release cells. Performed an inspection of the        flask to ensure all cells have detached. Placed “TIL Suspension”        3000 mL collection bag on dry wipes on a flat surface. NOTE:        Contacted area management if cells did not detach.    -   8.12.14 Initiated collection of TIL. Tilted the flask to ensure        hose is at the edge of the flask. NOTE: If the cell collection        hose was not at the junction of the wall and bottom membrane,        rapping the flask while tilted at a 450 angle is usually        sufficient to properly position the hose.    -   8.12.15 TIL Harvest. Released all clamps leading to the TIL        suspension collection bag.    -   8.12.16 TIL Harvest. Using the GatheRex, transferred the TIL        suspension into the 3000 mL collection bag. NOTE: Maintained the        tilted edge until all cells and media were collected.    -   8.12.17 TIL Harvest. Inspect membrane for adherent cells.    -   8.12.18 Rinsed flask membrane. Rinsed the bottom of the        G-Rex500MCS. Covered ˜¼ of gas exchange membrane with rinse        media.    -   8.12.19 Close clamps on G-Rex500MCS. Ensure all clamps are        closed.    -   8.12.20 Heat sealed. Heat seal (per Process Note 5.12) the        collection bag containing the TIL as close to the weld as        possible so that the overall tubing length remained        approximately the same.    -   8.12.21 Heat Sealed. Heat sealed (per Process Note 5.12) the        Supernatant bag.    -   8.12.22 Completed harvest of remaining G-Rex 500 MCS flasks.        Repeat Steps 8.12.2 and 8.12.5-8.12.21, pooling all TIL into the        same collection bag.    -   NOTE: IT WAS NECESSARY TO REPLACE THE 10 L SUPERNATANT BAG AFTER        EVERY 2ND FLASK. NOTE: Reference Process Note 5.16 for use of        multiple GatheRex devices.    -   8.12.23 Prepared LOVO source bag. Obtained a new 3000 mL        collection bag. Labeled as “LOVO Source Bag”, lot number, and        Initial/Date.    -   8.12.24 Prepared LOVO source bag. Heat sealed (per Process Note        5.12) the tubing on the “LOVO Source bag”, removing the female        luers, leaving enough line to weld.    -   8.12.25 Weighed LOVO Source Bag. Placed an appropriately sized        plastic bin on the scale and tare. Place the LOVO Source Bag,        including ports and lines, in the bin and record the dry weight    -   8.12.26 Transferred cell suspension into LOVO source bag. Closed        all clamps of a 170 μm gravity blood filter.    -   8.12.27 Transferred cell suspension into LOVO source bag.        Sterile welded (per Process Note 5.11) the long terminal end of        the gravity blood filter to the LOVO source bag.    -   8.12.28 Transferred cell suspension into LOVO source bag.        Sterile welded (per Process Note 5.11) one of the two source        lines of the filter to “pooled TIL suspension” collection bag.    -   8.12.29 Transferred cell suspension into LOVO source bag. Once        weld was complete, heat sealed (per Process Note 5.12) the        unused line on the filter to remove it.    -   8.12.30 Transferred cell suspension into LOVO source bag. Opened        all necessary clamps and elevate the TIL suspension by hanging        the collection bag on an IV pole to initiate gravity-flow        transfer of TIL through the blood filter and into the LOVO        source bag. Gently rotated or knead the TIL Suspension bag while        draining in order to keep the TIL in even suspension.    -   Note: Did not allow the LOVO source bag to hang from the        filtration apparatus. Laid LOVO source bag on dry wipes on a        flat surface.    -   8.12.31 Closed all clamps. Once all TIL were transferred to the        LOVO source bag, closed all clamps.    -   8.12.32 Heat Sealed. Heat sealed (per Process Note 5.12) as        close to weld as possible to remove gravity blood filter.    -   8.12.33 Removed Cell Counts Samples. In the BSC, using separate        3 mL syringes for each sample, removed 4×1.0 mL cell count        samples from the LOVO source bag using the needless injection        port. Placed samples in the cryovials prepared in Step 8.11.36.        NOTE: Wiped needless injection port with an alcohol pad and mix        LOVO source bag between each sample.    -   8.12.34 Performed Cell Counts. Performed cell counts and        calculations utilizing NC-200 and Process Note 5.14. Diluted        cell count samples initially by adding 0.5 mL of cell suspension        into 4.5 mL of AIM-V media prepared in Step 8.11.35. This gave a        1:10 dilution.    -   8.12.35 Recorded Cell Count sample volumes.    -   8.12.36 Determined Multiplication Factor

Parameter Formula Result Total cell count 8.12.35A + 8.12.35B C. μlsample Volume Multiplication C + 8.12.35A D. Factor

-   -   8.12.37 Selected protocols and enter multiplication factors.        Ensured the “Viable Cell Count Assay” protocol had been        selected, all multiplication factors, and sample and diluent        volumes had been entered. NOTE: If no dilution needed, enter        “Sample [μL]”=200, “Dilution [μL]”=0    -   8.12.38 Record Cell Counts from Nucleoview    -   8.12.39 Determined the Average Viability, Viable Cell        Concentration, and Total Nucleated Cell concentration of the        cell counts performed.

Parameter Formula Result Viability (8.12.38A + G. 8.12.38B) ÷ 2 ViableCell (8.12.38C + H. cells/mL Concentration 8.12.380) ÷ 2 Total NucleatedCell (8.12.38E + I. cells/mL Concentration 8.12.38F) ÷ 2

-   -   8.12.40 Determined Upper and Lower Limit for counts

Parameter Formula Result Lower Limit 8.12.39H × 0.9 J. cells/mL UpperLimit 8.12.39I × 1.1 K. cells/mL

-   -   8.12.41 Were both counts within acceptable limits?

Parameter Formula Result (Yes/No) Lower Limit 8.12.38 C and D ≥ 8.12.40JUpper Limit 8.12.38 C and D ≤ 8.12.40K NOTE: If either result was “No”performed second set of counts in steps 8.12.42-8.12.49.

-   -   8.12.42 If Applicable: Performed cell counts. Performed cell        counts and calculations in utilizing NC-200 and Process Note        5.14. NOTE: Dilution may be adjusted according based off the        expected concentration of cells.    -   8.12.43 If Applicable: Recorded Cell Count sample volumes    -   8.12.44 If Applicable: Determined Multiplication Factor

Parameter Formula Result Total cell count 8.12.43A + 8.12.43B C. μLsample Volume Multiplication C + 8.12.43A D. Factor

-   -   8.12.45 If Applicable: Selected protocols and enter        multiplication factors. Ensure the “Viable Cell Count Assay”        protocol had been selected, all multiplication factors, and        sample and diluent volumes had been entered.    -   NOTE: If no dilution needed, enter “Sample [μL]”=200, “Dilution        [μL]”=0    -   8.12.46 If Applicable: Record Cell Counts from Nucleoview    -   8.12.47 If Applicable: Determine the Average Viability, Viable        Cell Concentration, and Total Nucleated Cell concentration of        the cell counts performed.

Parameter Formula Result Viability (8.12.46A + G. 8.12.46B) ÷ 2 ViableCell (8.12.46C + H. cells/mL Concentration 8.12.460) ÷ 2 Total NucleatedCell (8.12.46E + I. cells/mL Concentration 8.12.46F) ÷ 2

-   -   8.12.48 If Applicable: Determined Upper and Lower Limit for        counts

Parameter Formula Result Lower Limit 8.12.47 H × 0.9 J. cells/mL UpperLimit 8.12.47 H × 1.1 K. cells/mL

-   -   8.12.49 If Applicable: Were counts within acceptable limits?

Parameter Formula Result (Yes/No) Lower Limit 8.12.46 C and D ≥ 8.12.48JUpper Limit 8.12.46 C and D > 8.12.48K NOTE: If either result was “No”continue to Step 8.12.50 to determine an average

-   -   8.12.50 If Applicable: Determined an average Viable Cell        Concentration and average Total Nucleated Cell Concentration        from all four counts performed.    -   8.12.51 QA Review of Cell Counts. QA personnel review        calculations performed in steps 8.12.38-8.12.50.    -   8.12.52 Weighed LOVO Source Bag. Placed an appropriately sized        plastic bin on the scale and tare. Placed the full LOVO source        bag in the bin and record the weight. Calculated the volume of        cell suspension.    -   8.12.53 Calculate Total Viable TIL Cells.

Parameter Formula Result Average Viable Cell 8.12.39 H* A. cells/mLConcentraion* or 8.12.47 H* or 8.12.50 E* Total Volume 8.12.52 C B. mLTotal Viable Cells A × B C. cells IS C ≥ 1.5 × 10⁹? Yes/No** *Circledstep reference used to determine Viable Cell Concentration. **If “Yes,”proceeded. If “No,” contacted Area Management.

-   -   8.12.54 Calculate Total Nucleated Cells.

Parameter Formula Result Average Total 8.12.39 I* A. cells/mL NucleatedCell or Concentraion* 8.12.47 I* or 8.12.50 J* Total Volume 8.5.52 C B.mL Total Nucleated A × B C. cells Cells *Circled step reference used todetermine Total Nucleated Cell Concentration.

-   -   8.12.55 Prepared Mycoplasma Diluent. In the BSC, removed 10.0 mL        from one supernatant bag via luer sample port and placed in a 15        mL conical. Label 15 mL conical “Mycoplasma Diluent” and keep in        the BSC for use in Step 8.14.69.    -   8.12.56 Review Section 8.12

8.13 LOVO

-   -   8.13.1 Turned on the LOVO using the switch on the back left of        the instrument. NOTE: Steps 8.13.1-8.13.13 may be performed        concurrently with Sections 8.11-8.12.    -   8.13.2 Checked weigh scales and pressure sensor.    -   8.13.3 Made sure there was nothing hanging on any of the weigh        scales and reviewed the reading for each scale. Recorded values        in Step 8.13.5. Note: If any of the scales read outside of a        range of 0+/−2 g, performed weigh scale calibration    -   8.13.4 If all scales were in tolerance with no weight hanging,        proceeded to hang a 1-kg weight on each scale (#1-4) and        reviewed the reading. Recorded Values in Step 8.13.5.    -   8.13.5 Scale Checked. Recorded the displayed values for each        scale. If values were in range, continue processing. If values        were not in range, perform Calibration.    -   8.13.6 Reviewed the pressure sensor reading on the Instrument        Operation Profile Screen and recorded. The acceptable range for        the pressure reading was 0+/−10 mmHg. If displayed value was out        of this range, stored a new atmospheric pressure setting, per        the machine instructions.    -   8.13.7 Repeated steps. If a new weigh scale calibration had been        performed or a new atmospheric pressure setting had been stored,        repeated Steps 8.13.3-8.13.6.    -   8.13.8 Started the “TIL G-Rex Harvest” protocol from the        drop-down menu.    -   8.13.9 The Solution 1 Screen displayed: Buffer type read        PlasmaLyte    -   8.13.10-8.13.16 Followed the LOVO touch screen prompts.    -   8.13.17 Determined the final product target volume.    -   NOTE: Using the total nucleated cells (TNC) value from Step        8.12.54 C and the chart below, determined the final product        target volume. Recorded the Final Product Volume (mL)

Final Product (Retentate) Volume to Cell Range Target (mL) 0 < Total(Viable + Dead) Cells ≤ 165 7.1 × 10¹⁰ 7.1 × 10¹⁰ < Total (Viable +Dead) Cells ≤ 215 1.1 × 10¹¹ 1.1 × 10¹¹ < Total (Viable + Dead) Cells ≤265 1.5 × 10¹¹ Note: If TVC from Step 8.12.53 C was > 1.5 × 10¹¹contacted Area Management.

-   -   8.13.18-8.13.22 Followed the LOVO touch screen prompts.    -   8.13.23 Loaded disposable kit. Prior to loading the disposable        kit, wipe pressure sensor port with an alcohol wipe followed by        a lint-free wipe. Load the disposable kit. Follow screen        directions on loading the disposable kit.    -   8.13.24 Removed filtrate bag. When the standard LOVO disposable        kit had been loaded, touched the Next button. The Container        Information and Location Screen displayed. Removed filtrate bag        from scale    -   8.13.25 Ensured Filtrate container was New and Off-Scale    -   8.13.26 Entered Filtrate capacity. Sterile welded a LOVO        Ancillary Bag onto the male luer line of the existing Filtrate        Bag. Ensured all clamps are open and fluid path is clear. Touch        the Filtrate Container Capacity entry field. A numeric keypad        displays. Enter the total new Filtrate capacity (5,000 mL).        Touch the button to accept the entry. NOTE: Estimated Filtrate        Volume should not exceed 5000 mL.    -   8.13.27 Placed Filtrate container on benchtop. NOTE: If tubing        was removed from the F clamp during welding, placed the tubing        back into the clamp. Placed the new Filtrate container on the        benchtop. DID NOT hang the Filtrate bag on weigh scale #3. Weigh        scale #3 will be empty during the procedure.    -   8.13.28 Followed the LOVO touch screen prompts after changes to        the filtrate container.    -   8.13.29 Ensured kit was loaded properly. The Disposable Kit Dry        Checks overlay displays. Checked that the kit was loaded        properly and all clamps were open. Checked all tubing for kinks        or other obstructions and correct if possible. Ensured kit was        properly installed and check all Robert's clamps. Pressed the        Yes button. All LOVO mechanical clamps closed automatically and        the Checking Disposable Kit Installation screen displays. The        LOVO went through a series of pressurizing steps to check the        kit.    -   8.13.30 Kit Check Results. If the Kit check passed, proceeded to        the next step. *If No, a second Kit Check could be performed        after checks have been complete. *If No, Checked all tubing for        kinks or other obstructions and correct *If No, Ensured kit was        properly installed and check all Robert's clamps. If the 2nd kit        check failed: Contact area management and prepare to        installation of new kit in Section 10.0. Repeat Step        8.13.23-Step 8.13.30 needed.    -   8.13.31 Attached PlasmaLyte. The Connect Solutions screen        displayed. The wash value would always be 3000 mL. Entered this        value on screen. Sterile welded the 3000 mL bag of PlasmaLyte to        the tubing passing through Clamp 1 per Process Note 5.11. Hung        the PlasmaLyte bag on an IV pole placing both corner bag loops        on the hook.    -   8.13.32 Verified that the PlasmaLyte was attached. Opened any        plastic clamps. Verified that the Solution Volume entry was 3000        mL. Touched the “Next” button. The Disposable Kit Prime overlay        displayed. Verified that the PlasmaLyte was attached and any        welds and plastic clamps on the tubing leading to the PlasmaLyte        bag were open, then touched the Yes button    -   8.13.33 Observed that the PlasmaLyte is moving. Disposable kit        prime starts and the Priming Disposable Kit Screen displays.        Visually observed that PlasmaLyte moving through the tubing        connected to the bag of PlasmaLyte. If no fluid was moving,        pressed the Pause Button on the screen and determined if a clamp        or weld was still closed. Once the problem had been solved,        pressed the Resume button on the screen to resume the Disposable        Kit Prime. When disposable kit prime finished successfully, the        Connect Source Screen displayed.    -   8.13.34-8.13.35 Followed the LOVO touch screen prompts.    -   8.13.36 Attached Source container to tubing. Sterile weld the        LOVO Source Bag prepared in Step 8.12.31 to the tubing passing        through Clamp S per Process Note 5.11. It could be necessary to        remove the tubing from the clamp. Note: Made sure to replace        source tubing into the S clamp if removed.    -   8.13.37 Hung Source container. Hung the Source container on the        IV pole placing both corner bag loops on the hook. DID NOT hang        the Source on weigh scale #1. Opened all clamps to the source        bag.    -   8.13.38 Verified Source container was attached. Touched the Next        button. The Source Prime overlay displayed. Verified that the        Source was attached to the disposable kit, and that any welds        and plastic clamps on the tubing leading to the Source were        open. Touched the Yes button.    -   8.13.39 Confirm PlasmaLyte was moving. Source prime started and        the Priming Source Screen displayed. Visually observed that        PlasmaLyte is moving through the tubing attached to the Source        bag. If no fluid is moving, press the Pause Button on the screen        and determine if a clamp or weld is still closed. Once the        problem was solved, pressed the Resume button on the screen to        resume the Source Prime.    -   8.13.40 Started Procedure Screen. When the Source prime finishes        successfully, the Start Procedure Screen displays. Pressed        Start, the “Pre-Wash Cycle 1” pause screen appears immediately        after pressing start.    -   8.13.41 Inverted In Process Bag. Removed the In Process Bag from        weigh scale #2 (can also remove tubing from the In Process top        port tubing guide) and manually invert it to allow the wash        buffer added during the disposable kit prime step to coat all        interior surfaces of the bag. Re-hang the In Process Bag on        weigh scale #2 (label on the bag was facing to the left).        Replace the top port tubing in the tubing guide, if it was        removed.    -   8.13.42 Inverted Source bag. Before pressing the Start button,        mixed the Source bag without removing it from the IV pole by        massaging the bag corners and gently agitating the cells to        create a homogeneous cell suspension. Pressed the Resume button.        The LOVO started processing fluid from the Source bag and the        Wash Cycle 1 Screen displays.    -   8.13.43 Source Rinse Pause. The Rinse Source Pause screen        displayed once the source container is drained and the LOVO had        added wash buffer to the Source bag. Without removing the Source        bag from IV pole, massaged the corners and mixed well. Pressed        Resume.    -   8.13.44 Mix In Process Bag Pause. To prepare cells for another        pass through the spinner, the In Process Bag was diluted with        wash buffer. After adding the wash buffer to the In Process Bag,        the LOVO pauses automatically and displays the “Mix In Process        Bag” Pause Screen. Without removing the bag from the weigh        scale, mixed the product well by gently squeezing the bag. Press        Resume.    -   8.13.45 Massage In Process Corners Pause. When the In Process        Bag was empty, wash buffer was added to the bottom port of the        In Process Bag to rinse the bag. After adding the rinse fluid,        the LOVO paused automatically and displayed the “Massage IP        corners” Pause Screen. When the “Massage IP corners” Pause        Screen displayed, DO NOT remove the bag from weigh scale #2.        With the In Process Bag still hanging on weigh scale #2, massage        the corners of the bag to bring any residual cells into        suspension. Ensured the bag was not swinging on the weigh scale        and pressed the Resume button.    -   8.13.46 Wait for Remove Products Screen. At the end of the LOVO        procedure, the Remove Products Screen displayed. When this        Screen displays, all bags on the LOVO kit could be manipulated.        Note: Did not touch any bags until the Remove Products        displayed.    -   8.13.47 Removed retentate bag. Placed a hemostat on the tubing        very close to the port on the Retentate bag to keep the cell        suspension from settling into the tubing. Heat sealed (per        Process Note 5.12) below the hemostat, making sure to maintain        enough line to weld in Step 8.13.48. Removed the retentate bag.    -   8.13.48 Prepared retentate bag for formulation. Welded (per        Process Note 5.11) the female luer lock end of a 4″ Plasma        Transfer Set to the retentate bag. Transferred the retentate bag        to the BSC for use in Step 8.14.11.    -   8.13.49 Removed Products. Followed the instructions on the        Remove Products Screen. Closed all clamps on the LOVO kit to        prevent fluid movement.    -   8.13.50 Removed Products. Touched the Next button. All LOVO        mechanical clamps opened and the Remove Kit Screen displayed.    -   8.13.51 Recorded Data. Followed the instructions on the Remove        Kit screen. Touched the “Next” button. All LOVO mechanical        clamps close and the Results Summary Screen displays. Recorded        the data from the results summary screen in table exactly as        they are displayed. Closed all pumps and filter support. Removed        the kit when prompted to do so by the LOVO.    -   *NOTE: All Times recorded were recorded directly from the LOVO        Results Summary Screen in HH:MM:SS format and (HH:MM:SS) format        when applicable    -   8.13.52-8.13.54 Protocol Selection through LOVO shutdown. Follow        the LOVO screen prompts.    -   8.13.55 Review Section 8.13

8.14 Final Formulation and Fill

-   -   8.14.1 Target volume/bag calculation. From the table below,        selected the number of CS750 bags to be filled, target fill        volume per bag, volume removed for retain per bag, and final        target volume per bag that corresponded to the Volume of LOVO        Retentate from Step 8.13.22.

Final Volume Volume Predicted Number Target Volume Final of of CS10Volume of of bags Fill removed Target LOVO to add to formulated to beVolume for retain Volume product product product filled per bag per bagper bag 165 mL 165 mL 330 mL 3 107 mL 7 mL 100 mL 215 mL 215 mL 430 mL 4105 mL 5 mL 100 mL 265 mL 265 mL 530 mL 4 130 mL 5 mL 125 mL

-   -   8.14.2 Prepared CRF Blank. Calculated volume of CS-10 and LOVO        wash buffer to formulate blank bag.

Final Target Volume per Bag Blank LOVO Wash Blank CS-10 8.14.1E BufferVolume Volume (mL) A B = A / 2 C = B mL mL mL

-   -   8.14.3 Prepared CRF Blank. Outside of the BSC, using the syringe        of LOVO Wash Buffer prepared in Step 8.11.29, added volume        calculated in Step 8.14.2 B to an empty CS750 bag via luer        connection. Note: Blank CS750 bag formulation does not need to        be done aseptically.    -   8.14.4 Prepared CRF Blank Using an appropriately sized syringe,        added the volume of CS-10 calculated in Step 8.14.2 to the same        CS750 bag prepared in Step 8.14.3. Placed a red cap on the CS750        bag.    -   8.14.5 Prepared CRF Blank. Removed as much air as possible from        the CS-750 bag as possible. Heat sealed (per Process Note 5.12)        the CS750 bag as close to the bag as possible, removing the        tubing.    -   8.14.6 Prepared CRF Blank.Label CS750 bag with “CRF Blank”, lot        number, and initial/date. Placed the CRF Blank on cold packs        until it was placed in the CRF.    -   8.14.7 Calculated required volume of IL-2. Calculated the volume        of IL-2 to add to the Final Product

Parameter Formula Result Final Retentate Volume Step 8.13.51 A. mL FinalFormulated Volume B = A × 2 B. mL Final IL-2 Concentration 300 IU/mL C.300 IU/mL desired (IU/mL) IU of IL-2 Required D = B × C D. IU IL-2Working Stock from 6 × 10⁴ IU/mL E. 6 × 10⁴ IU/mL Step 8.11.33 Volume ofIL-2 to Add to F = D ÷ E F. mL Final Product

-   -   8.14.8 Assembled Connect apparatus. Sterile welded (per Process        Note 5.11) a 4S-4M60 to a CC2 Cell Connect replacing a single        spike of the Cell Connect apparatus (B) with the 4-spike end of        the 4S-4M60 manifold at (G).    -   8.14.9 Assembled Connected apparatus. Sterile welded (per        Process Note 5.11) the CS750 Cryobags to the harness prepared in        Step 8.14.8, replacing one of the four male luer ends (E) with        each bag. Reference Step 8.14.1 to determine the number of bags        needed.    -   8.14.10 Assembled Connected apparatus. Welded (per Process Note        5.11) CS-10 bags to spikes of the 4S-4M60. Kept CS-10 cold by        placing the bags between two cold packs conditioned at 2-8° C.    -   8.14.11 Passed materials into the BSC.

Item # or Step Item Reference Quantity 4″ plasma transfer set 1 IL-2(6.0 × 10⁴) aliquot 8.11.33 1 Appropriate size syringe to add IL28.14.7F 1 LOVO retentate bag 8.13.48 1 Red Caps 5

-   -   8.14.12 Prepared TIL with IL-2. Using an appropriately sized        syringe, removed amount of IL-2 determined in Step 8.14.7 from        the “IL-2 6×10⁴” aliquot.    -   8.14.13 Prepared TIL with IL-2. Connect the syringe to the        retentate bag prepared in Step 8.13.48 via the Luer connection        and inject IL-2. 8.14.14 Prepare TIL with IL-2 Clear the line by        pushing air from the syringe through the line.    -   8.14.15 Labeled Formulated TIL Bag. Closed the clamp on the        transfer set and label bag as “Formulated TIL” and passed the        bag out of the BSC.    -   8.14.16 If applicable: Sample per sample plan. If there was        remaining “IL-2 6×10⁴” aliquot prepared in step 8.11.33, remove        a ˜5 mL sample retain according to the sample plan using an        appropriately sized syringe and dispense into a 50 mL conical        tube.    -   8.14.17 If applicable: Sampled per sample plan. Labeled with        sample plan inventory label and stored at 2-8° C. until        submitted to Login for testing per Sample Plan.    -   8.14.18 If applicable: Sampled per sample plan. Ensured that        LIMS sample plan sheet was filled out for removal of the sample.    -   8.14.19 Added the Formulated TIL bag to the apparatus. Once IL-2        had been added, welded (per Process Note 5.11) the “Formulated        TIL” bag to the remaining spike (A) on the apparatus prepared in        Step 8.14.10.    -   8.14.20 Added CS10. Passed the assembled apparatus with attached        Formulated TIL, CS-750 bags, and CS-10 into the BSC. NOTE: The        CS-10 bag and all CS-750 bags were placed between two cold packs        preconditioned at 2-8° C. Did not place Formulated TIL bag on        cold packs.    -   8.14.21 Added CS10. Ensured all clamps were closed on the        apparatus. Turn the stopcock so the syringe was closed.    -   8.14.22 Switched Syringes. Drew ˜10 mL of air into a 100 mL        syringe and replaced the 60 mL syringe on the apparatus.    -   8.14.23 Added CS10. Turned stopcock so that the line to the        CS750 bags is closed. Open clamps to the CS-10 bags and pull        volume calculated in Step 8.14.1B into syringe. NOTE: Multiple        syringes will be used to add appropriate volume of CS-10. NOTE:        Record volume from each syringe in Step 8.14.26    -   8.14.24 Added CS10. Closed clamps to CS-10 and open clamps to        the Formulated TIL bag and add the CS-10. Note: Add first 10.0        mL of CS10 at approximately 10.0 mL/minute. Add remaining CS-10        at approximate rate of 1.0 mL/sec. Note: Multiple syringes were        used to add appropriate volume of CS-10. Did not reuse a syringe        once it had been dispensed.    -   8.14.25 Added CS10. Recorded time. NOTE: The target time from        first addition of CS-10 to beginning of freeze is 30    -   8.14.26 Added CS10. Recorded the volume of each CS10 addition        and the total volume added. Total volume match calculated volume        from Step 8.14.1B    -   8.14.27 Added CS10. Closed all clamps to the CS10 bags.    -   8.14.28 Prepared CS-750 bags. Turned the stopcock so that the        syringe was open. Opened clamps to the Formulated TIL bag and        drew up suspension stopping just before the suspension reaches        the stopcock.    -   8.14.29 Prepared CS-750 bags. Closed clamps to the formulated        TIL bag. Turned stopcock so that it was open to the empty CS750        final product bags.    -   8.14.30 Prepared CS-750 bags. Using a new syringe, removed as        much air as possible from the CS750 final product bags by        drawing the air out. While maintaining pressure on the syringe        plunger, clamped the bags shut.    -   8.14.31 Prepared CS-750 bags. Draw ˜20 mL air into anew 100 mL        syringe and connect to the apparatus.    -   NOTE: Each CS-750 final product bag should be between two cold        packs to keep formulated TIL suspension cold.    -   8.14.32 Dispensed cells. Turned the stopcock so the line to the        final product bags was closed. Pulled the volume calculated in        Step 8.14.1 from the Formulated TIL bag into the syringe. NOTE:        Multiple syringes could be used to obtain correct volume.    -   8.14.33 Dispensed cells. Turned the stopcock so the line to the        formulated TIL bag is closed. Working with one final product bag        at a time, dispense cells into a final product bag. Recorded        volume of cells added to each CS750 bag in Step 8.14.35    -   8.14.34 Dispensed cells. Cleared the line with air from the        syringe so that the cells are even with the top of the spike        port. Closed the clamp on the filled bag. Repeated Step        8.14.29-Step 8.14.34 for each final product bag, gently mixing        formulated TIL bag between each.    -   8.14.35 Dispensed cells. Record volume of TIL placed in each        final product bag below.    -   8.14.36 Removed air from final product bags and take retain.        Once the last final product bag was filled, closed all clamps.    -   8.14.37 Removed air from final product bags and take retain.        Drew 10 mL of air into a new 100 mL syringe and replace the        syringe on the apparatus.    -   8.14.38 Removed air from final product bags and take retain.        Manipulating a single bag at a time, drew all of the air from        each product bag plus the volume of product for retain        determined in Step 8.14.1 D. NOTE: Upon removal of sample        volume, inverted the syringe and used air to clear the line to        the top port of the product bag. Clamped the line to the bag        once the retain volume and air was removed.    -   8.14.39 Recorded Volume Removed. Recorded volume of retain        removed from each bag.    -   8.14.40 Dispensed Retain. Dispensed retain into a 50 mL conical        tube and label tube as “Retain” and lot number. Repeat Step        8.14.37-Step 8.14.39 for each bag.    -   8.14.41 Prepared final product for cryopreservation. With a        hemostat, clamped the lines close to the bags. Removed syringe        and red cap luer connection on the apparatus that the syringe        was on. Passed apparatus out of the BSC.    -   8.14.42 Prepared final product for cryopreservation. Heat sealed        (per Process Note 5.12) at F, removing the empty retentate bag        and the CS-10 bags.    -   NOTE: Retained luer connection for syringe on the apparatus.        Disposed of empty retentate and CS-10 Bags.    -   8.14.43 Performed visual inspection. NOTE: Step 8.14.43—Step        8.14.46 may be performed concurrently with Step 8.14.47-Step        8.14.68. 8.14.44 Final Product Label Sample. Labeled final        product bags. Attached sample final product label below.    -   8.14.45 Prepared final product for cryopreservation. Held the        cryobags on cold pack or at 2-8° C. until cryopreservation.    -   8.14.46 Prepared external labels. Ensured the QA issued external        labels that will be attached to the cassettes labels match        corresponding final product label. Attached QA issued external        labels to cassettes. Attached a sample external label below:    -   8.14.47 Removed Cell Count Sample. Using an appropriately sized        pipette, remove 2.0 mL of retain removed in Step 8.14.38 and        place in a 15 mL conical tube to be used for cell counts.    -   8.14.48 Performed Cell Counts. Performed cell counts and        calculations utilizing the NC-200 per SOP-00314 and Process Note        5.14. NOTE: Diluted only one sample to appropriate dilution to        verify dilution is sufficient. Diluted additional samples to        appropriate dilution factor and proceed with counts.    -   8.14.49 Recorded Cell Count sample volumes. NOTE: If no dilution        needed, “Sample [μL]”=200, “Dilution [μL]”=0    -   8.14.50 Determined Multiplication Factor

Parameter Formula Result Total cell count 8.14.49A + 8.14.49B C. μLsample Volume Multiplication C ÷ 8.14.49A D. Factor

-   -   8.14.51 Select protocols and enter multiplication factors.        Ensure the “Viable Cell Count Assay” protocol has been selected,        all multiplication factors, and sample and diluent volumes have        been entered per SOP—00314    -   NOTE: If no dilution needed, enter “Sample [μL]”=200, “Dilution        [μL]”=0    -   8.14.52 Recorded File Name, Viability and Cell Counts from        Nucleoview.    -   8.14.53 Determined the Average of Viable Cell Concentration and        Viability of the cell counts performed.

Parameter Formula Result Viability (8.14.52A + E. % 8.14.52B) ÷ 2 ViableCell (8.14.52C + F. cells/mL Concentration 8.14.52D) ÷ 2

-   -   8.14.54 Determined Upper and Lower Limit for counts.

Parameter Formula Result Lower Limit 8.14.53F × 0.9 G. cells/mL UpperLimit 8.14.53F × 1.1 H. cells/mL

-   -   8.14.55 Were both counts within acceptable limits?

Parameter Formula Result (Yes/No) Lower Limit 8.14.52 C and D ≥ 8.14.54GUpper Limit 8.14.52 C and D ≤ 8.14.54H NOTE: If either result is “No”perform second set of counts in steps 8.14.56-8.14.63

-   -   8.14.56 If Applicable: Performed cell counts. Performed cell        counts and calculations in utilizing NC-200 per SOP-00314 and        Process Note 5.14.    -   NOTE: Dilution may be adjusted according based off the expected        concentration of cells.    -   8.14.57 If Applicable: Recorded Cell Count sample volumes.    -   8.14.58 If Applicable: Determined Multiplication Factor

Parameter Formula Result Total cell count 8.14.57A + 8.14.57B C. mLsample Volume Multiplication C ÷ 8.14.57A D. Factor

-   -   8.14.59 If Applicable: Selected protocols and entered        multiplication factors. Ensured the “Viable Cell Count Assay”        protocol had been selected, all multiplication factors, and        sample and diluent volumes had been entered.    -   NOTE: If no dilution needed, enter “Sample [μL]”=200, “Dilution        [μL]”=0    -   8.14.60 If Applicable: Recorded Cell Counts from Nucleoview    -   8.14.61 If Applicable: Determined the Average of Viable Cell        Concentration and Viability of the cell counts performed.

Parameter Formula Result Viability (8.14.60A + E. % 8.14.60B) ÷ 2 ViableCell (8.14.60C + F. cells/mL Concentration 8.14.60D) ÷ 2

-   -   8.14.62 If Applicable: Determined Upper and Lower Limit for        counts.

Parameter Formula Result Lower Limit 8.14.61F × 0.9 G. cells/mL UpperLimit 8.14.61F × 1.1 H. cells/mL

-   -   8.14.63 If Applicable: Were counts within acceptable limits?

Parameter Formula Result (Yes/No) Lower Limit 8.14.60 C and D ≥ 8.14.62GUpper Limit 8.14.60 C and D ≤ 8.14.62H NOTE: If either result is “No”continue to Step 8.14.64 to determine an average.

-   -   8.14.64 If Applicable: Determined an average Viable Cell        Concentration from all four counts performed.    -   8.14.65 Calculated Flow. Cytometry Sample. Performed calculation        to ensure sufficient cell concentration for flow cytometry        sampling.

Viable Cell Concentration From Step 8.14.53 F* Or Step 8.14.61 F* OrTarget Volume Required Step 8.14.64 E* for 6 × 10⁷ TVC Is B ≤ 1.0 mL? AB = 6 × 10⁷ cells/ A (Yes/No**) cells/mL mL *Circle step reference usedto determine Viable Cell Concentration **NOTE: If “No”, contact areamanagement.

-   -   8.14.66 Calculated IFN-γ. Sample Performed calculation to ensure        sufficient cell concentration for IFN-γ sampling.

Viable Cell Concentration From Step 8.14.53 F* Or Step 8.14.61 F* OrVolume Required for Step 8.14.64 E* Minimum 1.5 × 10⁷ TVC Is B ≤ 1.0 mL?A B = 1.5 × 10⁷ cells / A (Yes/No**) cells/mL mL *Circle step referenceused to determine Viable Cell Concentration **NOTE: If “No”, contactarea management.

-   -   8.14.67 Reported Results. Completed forms for submission with        samples.    -   8.14.68 Heat Sealed. Once sample volumes had been determined,        heat sealed (per Process Note 5.12) Final Product Bags as close        to the bags as possible to remove from the apparatus.    -   8.14.69 Labeled and Collected Samples per Sample Plan.

Sample Volume to Number of Add to Container Sample Containers Each TypeDestination *Mycoplasma 1 1.0 mL 15 mL Login Conical Endotoxin 2 1.0 mL 2 mL Cryovial Login Gram Stain 1 1.0 mL  2 mL Cryovial Login IFN-g 11.0 mL  2 mL Cryovial Login Flow 1 1.0 mL  2 mL Cryovial Login Cytometry**Bac-T 2 1.0 mL Bac-T Bottle Login Sterility QC Retain 4 1.0 mL  2 mLCryovial CRF Satellite Vials 10 0.5 mL  2 mL Cryovial CRF *NOTE: For theMycoplasma sample, add formulated cell suspension volume to the 15 mLconical labelled “Mycoplasma Diluent” from Step 8.12.55. **NOTE: Proceedto Step 8.14.70 for Bac-T inoculation.

-   -   8.14.70 Sterility & BacT. Testing Sampling. In the BSC, remove a        1.0 mL sample from the retained cell suspension collected in        Step 8.14.38 using an appropriately sized syringe and inoculate        the anaerobic bottle. Repeat the above for the aerobic bottle.        NOTE: Store Bac-T bottles are room temperature and protect from        light.    -   8.14.71 Labeled and stored samples. Labeled all samples with        sample plan inventory labels and store appropriately until        transfer to Login. NOTE: Proceeded to Section 8.15 for        cryopreservation of final product and samples.    -   8.14.72 Signed for sampling. Ensured that LIMS sample plan sheet        is completed for removal of the samples.    -   8.14.73 Sample Submission. Submitted all Day 22 testing samples        to Login.    -   8.14.74 Environmental Monitoring. After processing, verified BSC        and personnel monitoring had been performed.    -   8.14.75 Review Section 8.14    -   8.15 Final Product Cryopreservation    -   8.15.1 Prepared Controlled Rate Freezer. Verified the CRF had        been set up prior to freeze. Record CRF Equipment.        Cryopreservation is performed.    -   8.15.2 Set up CRF probes. Punctured the septum on the CRF blank        bag. Inserted the 6 mL vial temperature probe.    -   8.15.3 Placed final product and samples in CRF. Placed blank bag        into preconditioned cassette and transferred into the        approximate middle of the CRF rack. Transferred final product        cassettes into CRF rack and vials into CRF vial rack.    -   8.15.4 Placed final product and samples in CRF. Transferred        product racks and vial racks into the CRF. Recorded the time        that the product is transferred into the CRF and the chamber        temperature in Step 8.15.5. NOTE: Evenly distributed the        cassettes and vial rack in the CRF, allowed as much space as        possible between each shelf.    -   8.15.5 Determined the time needed to reach 4° C.±1.5° C. and        proceed with the CRF run. Once the chamber temperature reached        4° C.±1.5° C., started the run. Recorded time.

Parameter Formula Value Time Final Product is transferred to CRF (HHMM)Temperature Final Product is transferred into From ° C. CRF monitor B.CRF Start Time (HHMM) Elapsed Time from Formulation to CRF Start C = B −Step min 8.14.25A

-   -   8.15.6 CRF Completed and Stored. Stopped the CRF after the        completion of the run. Remove cassettes and vials from CRF.        Transferred cassettes and vials to vapor phase LN2 for storage.        Recorded storage location

Post Processing Summary

-   -   Post-Processing: Final Drug Product    -   (Day 22) Determination of CD3+ Cells on Day 22 REP by Flow        Cytometry    -   (Day 22) Gram Staining Method (GMP)    -   (Day 22) Bacterial Endotoxin Test by Gel Clot LAL Assay (GMP)    -   (Day 16) BacT Sterility Assay (GMP)    -   (Day 16) Mycoplasma DNA Detection by TD-PCR (GMP)    -   Acceptable Appearance Attributes (Step 8.14.43)    -   (Day 22) BacT Sterility Assay (GMP)    -   (Day 22) IFN-gamma Assay

Example 2. Genetic Editing of a Cryopreserved TIL Therapy Using TALENucleases

This example describes the use of a genetic editing step in conjunctionwith a TIL manufacturing process, such as the process shown in FIG. 20.

Optimization of human TIL electroporation is performed as follows.Four-6 pre-REP TIL lines derived from melanoma will be identified thatexpress >25% PD1. Selected TIL lines will be thawed, rested, andactivated, working 1 line at a time. Five-million activated TILs will beelectroporated with each of the following control or test RNA: noElectroporation control (NE); no RNA control; green fluorescent protein(GFP) mRNA transfection control, CD52 TALEN KO control, PD1 TALEN, LAG-3TALEN, and TIM-3 TALEN. Viable cells will be counted and an aliquot setaside for transfection efficiency measurements (GFP expression).Electroporated cells will be expanded by REP. Post-REP TILs will beassessed for cell viability and fold expansion (cell counter) and targetgene knockdown (flow and qPCR). The target results are >80% viability orwithin 10% of NE; >70% transfection; fold expansion of TILs to within30% of NE; and >50% knockout.

Implementation of TALEN-mediated PD-1 knockout to the TIL manufacturingprocess is performed as follows. Up to 6 fresh tumors from severalhistologies that may include melanoma, sarcoma, and breast and lungcancer will be used. Research-scale preps will be performed according tothe processes shown in FIGS. 20 and 21. Electroporation conditions fromprior experiments will be applied at pre-determined time points of eachof the processes. Post-REP TILs will be assessed for: Cell viability andfold expansion (cell counter); Cell phenotypes (flow cytometry); TCR Vβrepertoire (flow); T cell effector functions (IFN-γ production uponre-stimulation); and target gene knockdown (flow and qPCR). The targetresults are: >80% viability or within 10% of NE; >70% transfection; foldexpansion within 30% of NE; T cell lineages/subsets maintained comparedto TILs produced by process 2A; adequate TIL potency; and >50% knockoutof PD-1. Following this work, one full-scale preparation of PD-1knockdown TILs will be carried out and fully characterized.

Validation of the silencing of two additional target genes in additionto PD-1 will also be tested.

Example 3. TALEN-Mediated Inactivation of PD-1 in TILs

This example describes TALEN-mediated inactivation of PD-1 inconjunction with a TIL manufacturing process described herein, such as aprocess shown in FIG. 20 or FIG. 21. The TIL manufacturing process mayinclude early addition of OKT-3 and/or a 4-1BB agonist to the cellculture medium e.g., as illustrated in Embodiment 2 of FIG. 21. TheOKT-3 and/or 4-1BB agonist may optionally be added beginning on Day 0 orDay 1 of the first expansion or the second expansion.

TALEN construction and electroporation may be carried out according tomethods described by Menger, et al., Cancer Res., 2016 Apr. 15;76(8):2087-93, Gautron, et al., et al., Molecular Therapy: Nucleic Acids2017, 9:312-321, or U.S. Pat. No. 9,458,439, the disclosures of each ofwhich are incorporated by reference herein. TALEN targeting the PD-1gene will be produced, e.g., using a solid phase assembly methoddescribed by Daboussi et al., Nat Commun 2014; 5:3831, which isincorporated by reference herein, or may be obtained commercially fromTrilink Biotechnologies or other providers as described in Gautron, etal., et al., Molecular Therapy: Nucleic Acids 2017, 9:312-321. Theprocedure described in Gautron, et al., et al., Molecular Therapy:Nucleic Acids 2017, 9:312-321, and described elsewhere herein, foractivation of TILs, may be employed, wherein TILs are activated usingDynabeads human T-cell activator CD3/CD28 beads (available commerciallyfrom Invitrogen) at ratio of 1:1 CD3⁺ bead:cell or human T-cellactivator CD3/CD28 antibody complexes such as Immunocult CD3/CD28activator (available commercially from StemCell Technologies). A totalof 5×10⁶ TILs/180 μL of BTX cytoporation medium T may be mixed withabout 10 to 20 μg of the in vitro transcribed TALEN mRNA (mMESSAGEmMACHINE T7 kit; Ambion), before electroporation using an Agile PulseBTX system (Harvard Apparatus). Electroporated cells may be expanded bypre-REP and REP methods described elsewhere herein. Post-REP TILs willbe assessed for cell viability and fold expansion (cell counter) andPD-1 knockdown (flow and qPCR). The target results are >80% viability orwithin 10% of a “no electroporation” (NE) control; >70% transfection;fold expansion of TILs to within 30% of NE; and >50% PD-1 knockout.Electroporation methods known in the art, such as those described inU.S. Pat. Nos. 6,010,613 and 6,078,490, the disclosures of which areincorporated by reference herein, may be used.

Pulsed electroporation optimization may be performed using methodsdescribed herein or as follows. A first set of experiments wereperformed on TILs in order to determine a voltage range in which cellscould be transfected. Five different programs were tested:

Group 1 Group 2 Group 3 Duration Interval Duration Interval DurationInterval Program Pulses V (ms) (ms) Pulses V (ms) (ms) Pulses V (ms)(ms) 1 1 600 0.1 0.2 1 600 0.1 100 4 130 0.2 2 2 1 900 0.1 0.2 1 900 0.1100 4 130 0.2 2 3 1 1200 0.1 0.2 1 1200 0.1 100 4 130 0.2 2 4 1 1200 0.110 1 900 0.1 100 4 130 0.2 2 5 1 900 0.1 20 1 600 0,1 100 4 130 0.2 2

TILs may be electroporated in 0.4 cm gap cuvette (on the order of about10⁶ cells/mL) with about 20 μg of plasmids encoding GFP and controlplasmids pUC using the different electroporation programs. About 24hours post electroporation, GFP expression was analyzed inelectroporated cells by flow cytometry to determine the efficiency oftransfection. The minimal voltage required for plasmid electroporationin TILs has been previously reported in International Patent ApplicationPublication No. WO 2014/184744 and U.S. Patent Application PublicationNo. US 2013/0315884 A1, the disclosures of which are incorporated byreference herein, and program 3 and 4 each allow for an efficient TALENincorporation process for TILs.

The TALEN construct shown in FIG. 23 targets exon 2 of the Pdcd1 gene,as shown, and may be used for the inactivation of PD1.

The examples set forth above are provided to give those of ordinaryskill in the art a complete disclosure and description of how to makeand use the embodiments of the compositions, systems and methods of theinvention, and are not intended to limit the scope of what the inventorsregard as their invention. Modifications of the above-described modesfor carrying out the invention that are obvious to persons of skill inthe art are intended to be within the scope of the following claims. Allpatents and publications mentioned in the specification are indicativeof the levels of skill of those skilled in the art to which theinvention pertains.

All headings and section designations are used for clarity and referencepurposes only and are not to be considered limiting in any way. Forexample, those of skill in the art will appreciate the usefulness ofcombining various aspects from different headings and sections asappropriate according to the spirit and scope of the invention describedherein.

All references cited herein are hereby incorporated by reference hereinin their entireties and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

Many modifications and variations of this application can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. The specific embodiments and examplesdescribed herein are offered by way of example only, and the applicationis to be limited only by the terms of the appended claims, along withthe full scope of equivalents to which the claims are entitled.

What is claimed is:
 1. A method for expanding tumor infiltratinglymphocytes (TILs) into a therapeutic population of TILs comprising: (a)obtaining a first population of TILs from a tumor resected from apatient by processing a tumor sample obtained from the patient intomultiple tumor fragments; (b) adding tumor fragments into a closedsystem, wherein the tumor fragments are from a tumor resected from apatient; (c) performing a first expansion by culturing the firstpopulation of TILs in a cell culture medium comprising IL-2, andoptionally OKT-3, to produce a second population of TILs, wherein thefirst expansion is performed in a closed container providing a firstgas-permeable surface area, wherein the first expansion is performed forabout 3-14 days to obtain the second population of TILs, wherein thesecond population of TILs is at least 50-fold greater in number than thefirst population of TILs, and wherein the transition from step (b) tostep (c) occurs without opening the system; (d) performing a secondexpansion by supplementing the cell culture medium of the secondpopulation of TILs with additional IL-2, optionally OKT-3, and antigenpresenting cells (APCs), to produce a third population of TILs, whereinthe second expansion is performed for about 7-14 days to obtain thethird population of TILs, wherein the third population of TILs is atherapeutic population of TILs, wherein the second expansion isperformed in a closed container providing a second gas-permeable surfacearea, and wherein the transition from step (c) to step (d) occurswithout opening the system; (e) harvesting the therapeutic population ofTILs obtained from step (d), wherein the transition from step (d) tostep (e) occurs without opening the system; (f) transferring theharvested TIL population from step (e) to an infusion bag, wherein thetransfer from step (e) to (f) occurs without opening the system; and (g)at any time during the method, gene-editing at least a portion of theTILs.
 2. The method according to claim 1, further comprising the step ofcryopreserving the infusion bag comprising the harvested TIL populationin step (f) using a cryopreservation process.
 3. The method according toclaim 1, wherein the cryopreservation process is performed using a 1:1ratio of harvested TIL population to cryopreservation media.
 4. Themethod according to claim 1, wherein the antigen-presenting cells areperipheral blood mononuclear cells (PBMCs).
 5. The method according toclaim 4, wherein the PBMCs are irradiated and allogeneic.
 6. The methodaccording to claim 4, wherein the PBMCs are added to the cell culture onany of days 9 through 14 in step (d).
 7. The method according to claim1, wherein the antigen-presenting cells are artificialantigen-presenting cells.
 8. The method according to claim 1, whereinthe harvesting in step (e) is performed using a membrane-based cellprocessing system.
 9. The method according to claim 1, wherein theharvesting in step (e) is performed using a LOVO cell processing system.10. The method according to claim 1, wherein the multiple fragmentscomprise about 4 to about 50 fragments, wherein each fragment has avolume of about 27 mm³.
 11. The method according to claim 1, wherein themultiple fragments comprise about 30 to about 60 fragments with a totalvolume of about 1300 mm³ to about 1500 mm³.
 12. The method according toclaim 9, wherein the multiple fragments comprise about 50 fragments witha total volume of about 1350 mm³.
 13. The method according to claim 1,wherein the multiple fragments comprise about 50 fragments with a totalmass of about 1 gram to about 1.5 grams.
 14. The method according toclaim 1, wherein the cell culture medium is provided in a containerselected from the group consisting of a G-container and a Xuri cellbag.15. The method according to claim 1, wherein the cell culture medium instep (d) further comprises IL-15 and/or IL-21.
 16. The method accordingto claim any of the preceding claims, wherein the IL-2 concentration isabout 10,000 IU/mL to about 5,000 IU/mL.
 17. The method according toclaim 15, wherein the IL-15 concentration is about 500 IU/mL to about100 IU/mL.
 18. The method according to claim 1, wherein the IL-21concentration is about 20 IU/mL to about 0.5 IU/mL.
 19. The methodaccording to claim 1, wherein the infusion bag in step (f) is aHypoThermosol-containing infusion bag.
 20. The method according to claim3, wherein the cryopreservation media comprises dimethlysulfoxide(DMSO).
 21. The method according to claim 17, wherein the wherein thecryopreservation media comprises 7% to 10% DMSO.
 22. The methodaccording to claim 1, wherein the first period in step (c) and thesecond period in step (e) are each individually performed within aperiod of 10 days, 11 days, or 12 days.
 23. The method according toclaim 1, wherein the first period in step (c) and the second period instep (e) are each individually performed within a period of 11 days. 24.The method according to claim 1, wherein steps (a) through (f) areperformed within a period of about 10 days to about 22 days.
 25. Themethod according to claim 1, wherein steps (a) through (f) are performedwithin a period of about 20 days to about 22 days.
 26. The methodaccording to claim 1, wherein steps (a) through (f) are performed withina period of about 15 days to about 20 days.
 27. The method according toclaim 1, wherein steps (a) through (f) are performed within a period ofabout 10 days to about 20 days.
 28. The method according to claim 1,wherein steps (a) through (f) are performed within a period of about 10days to about 15 days.
 29. The method according to claim 1, whereinsteps (a) through (f) are performed in 22 days or less.
 30. The methodaccording to claim 1, wherein steps (a) through (f) are performed in 20days or less.
 31. The method according to claim 1, wherein steps (a)through (f) are performed in 15 days or less.
 32. The method accordingto claim 1, wherein steps (a) through (f) are performed in 10 days orless.
 33. The method according to claim 2, wherein steps (a) through (f)and cryopreservation are performed in 22 days or less.
 34. The methodaccording to any one of claims 1 to 33, wherein the therapeuticpopulation of TILs harvested in step (e) comprises sufficient TILs for atherapeutically effective dosage of the TILs.
 35. The method accordingto claim 34, wherein the number of TILs sufficient for a therapeuticallyeffective dosage is from about 2.3×10¹⁰ to about 13.7×10¹⁰.
 36. Themethod according to any one of claims 1 to 35, wherein steps (b) through(e) are performed in a single container, wherein performing steps (b)through (e) in a single container results in an increase in TIL yieldper resected tumor as compared to performing steps (b) through (e) inmore than one container.
 37. The method according to any one of claims 1to 36, wherein the antigen-presenting cells are added to the TILs duringthe second period in step (d) without opening the system.
 38. The methodaccording to any one of claims 1 to 37, wherein the third population ofTILs in step (d) provides for increased efficacy, increasedinterferon-gamma production, increased polyclonality, increased averageIP-10, and/or increased average MCP-1 when administered to a subject.39. The method according to any one of claims 1 to 38, wherein the thirdpopulation of TILs in step (d) provides for at least a five-fold or moreinterferon-gamma production when administered to a subject.
 40. Themethod according to any one of claims 1 to 39, wherein the thirdpopulation of TILs in step (d) is a therapeutic population of TILs whichcomprises an increased subpopulation of effector T cells and/or centralmemory T cells relative to the second population of TILs, wherein theeffector T cells and/or central memory T cells in the therapeuticpopulation of TILs exhibit one or more characteristics selected from thegroup consisting of expressing CD27+, expressing CD28+, longertelomeres, increased CD57 expression, and decreased CD56 expressionrelative to effector T cells, and/or central memory T cells obtainedfrom the second population of cells.
 41. The method according to any oneof claims 1 to 40, wherein the effector T cells and/or central memory Tcells obtained from the third population of TILs exhibit increased CD57expression and decreased CD56 expression relative to effector T cellsand/or central memory T cells obtained from the second population ofcells.
 42. The method according to any one of claims 1 to 41, whereinthe risk of microbial contamination is reduced as compared to an opensystem.
 43. The method according to any one of claims 1 to 42, whereinthe TILs from step (g) are infused into a patient.
 44. The methodaccording to any one of claims 1 to 43, wherein the multiple fragmentscomprise about 4 fragments.
 45. The method according to any one ofclaims 1 to 44, wherein the cell culture medium further comprises a4-1BB agonist and/or an OX40 agonist during the first expansion, thesecond expansion, or both.
 46. The method according to claim 45, whereinand the gene-editing is carried out after the 4-1BB agonist and/or theOX40 agonist is introduced into the cell culture medium.
 47. The methodaccording to claim 45, wherein and the gene-editing is carried outbefore the 4-1BB agonist and/or the OX40 agonist is introduced into thecell culture medium.
 48. The method according to any of claims 1-47,wherein the gene-editing is carried out on TILs from one or more of thefirst population, the second population, and the third population. 49.The method according to any of claims 1-47, wherein the gene-editing iscarried out on TILs from the first expansion, or TILs from the secondexpansion, or both.
 50. The method according to any of claims 1-47,wherein the gene-editing is carried out after the first expansion andbefore the second expansion.
 51. The method according to any of claims1-47, wherein the gene-editing is carried out before step (c), beforestep (d), or before step (e).
 52. The method according to any of claims1-47, wherein the cell culture medium comprises OKT-3 during the firstexpansion and/or during the second expansion, and the gene-editing iscarried out before the OKT-3 is introduced into the cell culture medium.53. The method according to any of claims 1-47, wherein the cell culturemedium comprises OKT-3 during the first expansion and/or during thesecond expansion, and the gene-editing is carried out after the OKT-3 isintroduced into the cell culture medium.
 54. The method according to anyof claims 1-47, wherein the cell culture medium comprises OKT-3beginning on the start day of the first expansion, and the gene-editingis carried out after the TILs have been exposed to the OKT-3.
 55. Themethod according to any of claims 1-54, wherein the gene-editing causesexpression of one or more immune checkpoint genes to be silenced orreduced in at least a portion of the therapeutic population of TILs. 56.The method according to claim 55, wherein said one or more immunecheckpoint genes is/are selected from the group comprising PD-1, CTLA-4,LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6,PTPN22, PDCD1, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9,CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD,FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB,HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF,GUCY1A2, GUCY1A3, GUCY1B2, and GUCY1B3.
 57. The method according toclaim 55, wherein said one or more immune checkpoint genes is/areselected from the group comprising PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3),Cish, TGFβ, and PKA.
 58. The method according to any of claims 1-54,wherein the gene-editing causes expression of one or more immunecheckpoint genes to be enhanced in at least a portion of the therapeuticpopulation of TILs, the immune checkpoint gene(s) being selected fromthe group comprising CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL-4,IL-7, IL-10, IL-15, IL-21, the NOTCH 1/2 intracellular domain (ICD),and/or the NOTCH ligand mDLL1.
 59. The method according to any of claims1-58, wherein the gene-editing comprises the use of a programmablenuclease that mediates the generation of a double-strand orsingle-strand break at said one or more immune checkpoint genes.
 60. Themethod according to any of claims 1-58, wherein the gene-editingcomprises one or more methods selected from a CRISPR method, a TALEmethod, a zinc finger method, and a combination thereof.
 61. The methodaccording to any of claims 1-58, wherein the gene-editing comprises aCRISPR method.
 62. The method according to claim 61, wherein the CRISPRmethod is a CRISPR/Cas9 method.
 63. The method according to any ofclaims 1-58, wherein the gene-editing comprises a TALE method.
 64. Themethod according to any of claims 1-58, wherein the gene-editingcomprises a zinc finger method.
 65. A method for treating a subject withcancer, the method comprising administering expanded tumor infiltratinglymphocytes (TILs) comprising: (a) obtaining a first population of TILsfrom a tumor resected from a subject by processing a tumor sampleobtained from the patient into multiple tumor fragments; (b) adding thetumor fragments into a closed system; (c) performing a first expansionby culturing the first population of TILs in a cell culture mediumcomprising IL-2, and optionally OKT-3, to produce a second population ofTILs, wherein the first expansion is performed in a closed containerproviding a first gas-permeable surface area, wherein the firstexpansion is performed for about 3-14 days to obtain the secondpopulation of TILs, wherein the second population of TILs is at least50-fold greater in number than the first population of TILs, and whereinthe transition from step (b) to step (c) occurs without opening thesystem; (d) performing a second expansion by supplementing the cellculture medium of the second population of TILs with additional IL-2,optionally OKT-3, and antigen presenting cells (APCs), to produce athird population of TILs, wherein the second expansion is performed forabout 7-14 days to obtain the third population of TILs, wherein thethird population of TILs is a therapeutic population of TILs, whereinthe second expansion is performed in a closed container providing asecond gas-permeable surface area, and wherein the transition from step(c) to step (d) occurs without opening the system; (e) harvesting thetherapeutic population of TILs obtained from step (d), wherein thetransition from step (d) to step (e) occurs without opening the system;and (f) transferring the harvested TIL population from step (e) to aninfusion bag, wherein the transfer from step (e) to (f) occurs withoutopening the system; (g) optionally cryopreserving the infusion bagcomprising the harvested TIL population from step (f) using acryopreservation process; (h) administering a therapeutically effectivedosage of the third population of TILs from the infusion bag in step (g)to the patient; and (i) at any time during the method steps (a)-(f),gene-editing at least a portion of the TILs.
 66. The method according toclaim 65, wherein the therapeutic population of TILs harvested in step(e) comprises sufficient TILs for administering a therapeuticallyeffective dosage of the TILs in step (h).
 67. The method according toclaim 66, wherein the number of TILs sufficient for administering atherapeutically effective dosage in step (h) is from about 2.3×10¹⁰ toabout 13.7×10¹⁰.
 68. The method according to claim 67, wherein theantigen presenting cells (APCs) are PBMCs.
 69. The method according toclaim 68, wherein the PBMCs are added to the cell culture on any of days9 through 14 in step (d).
 70. The method according to any of claims 65to 69, wherein prior to administering a therapeutically effective dosageof TIL cells in step (h), a non-myeloablative lymphodepletion regimenhas been administered to the patient.
 71. The method according to claim70, where the non-myeloablative lymphodepletion regimen comprises thesteps of administration of cyclophosphamide at a dose of 60 mg/m²/dayfor two days followed by administration of fludarabine at a dose of 25mg/m²/day for five days.
 72. The method according to any of claims 65 to71, further comprising the step of treating the patient with a high-doseIL-2 regimen starting on the day after administration of the TIL cellsto the patient in step (h).
 73. The method according to claim 72,wherein the high-dose IL-2 regimen comprises 600,000 or 720,000 IU/kgadministered as a 15-minute bolus intravenous infusion every eight hoursuntil tolerance.
 74. The method according to any of claims 65-73,wherein the third population of TILs in step (d) is a therapeuticpopulation of TILs which comprises an increased subpopulation ofeffector T cells and/or central memory T cells relative to the secondpopulation of TILs, wherein the effector T cells and/or central memory Tcells in the therapeutic population of TILs exhibit one or morecharacteristics selected from the group consisting of expressing CD27+,expressing CD28+, longer telomeres, increased CD57 expression, anddecreased CD56 expression relative to effector T cells, and/or centralmemory T cells obtained from the second population of cells.
 75. Themethod according to any of claims 65-74, wherein the effector T cellsand/or central memory T cells in the therapeutic population of TILsexhibit increased CD57 expression and decreased CD56 expression relativeto effector T cells and/or central memory T cells obtained from thesecond population of cells.
 76. The method according to any of claims65-75, wherein the cancer is selected from the group consisting ofmelanoma, ovarian cancer, cervical cancer, non-small-cell lung cancer(NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused byhuman papilloma virus, head and neck cancer (including head and necksquamous cell carcinoma (HNSCC)), renal cancer, and renal cellcarcinoma.
 77. The method according to any of claims 65-76, wherein thecancer is selected from the group consisting of melanoma, HNSCC,cervical cancers, and NSCLC.
 78. The method according to any of claims65-77, wherein the cancer is melanoma.
 79. The method according to anyof claims 65-78, wherein the cancer is HNSCC.
 80. The method accordingto any of claims 65-79, wherein the cancer is a cervical cancer.
 81. Themethod according to any of claims 65-80, wherein the cancer is NSCLC.82. The method according to any one of claims 65 to 81, wherein the cellculture medium further comprises a 4-1BB agonist and/or an OX40 agonistduring the first expansion, the second expansion, or both.
 83. Themethod according to claim 82, wherein and the gene-editing is carriedout after the 4-1BB agonist and/or the OX40 agonist is introduced intothe cell culture medium.
 84. The method according to claim 82, whereinand the gene-editing is carried out before the 4-1BB agonist and/or theOX40 agonist is introduced into the cell culture medium.
 85. The methodaccording to any of claims 65-84, wherein the gene-editing is carriedout on TILs from one or more of the first population, the secondpopulation, and the third population.
 86. The method according to any ofclaims 65-84, wherein the gene-editing is carried out on TILs from thefirst expansion, or TILs from the second expansion, or both.
 87. Themethod according to any of claims 65-84, wherein the gene-editing iscarried out after the first expansion and before the second expansion.88. The method according to any of claims 65-84, wherein thegene-editing is carried out before step (c), before step (d), or beforestep (e).
 89. The method according to any of claims 65-88, wherein thecell culture medium comprises OKT-3 during the first expansion and/orduring the second expansion, and the gene-editing is carried out beforethe OKT-3 is introduced into the cell culture medium.
 90. The methodaccording to any of claims 65-88, wherein the cell culture mediumcomprises OKT-3 during the first expansion and/or during the secondexpansion, and the gene-editing is carried out after the OKT-3 isintroduced into the cell culture medium.
 91. The method according to anyof claims 65-88, wherein the cell culture medium comprises OKT-3beginning on the start day of the first expansion, and the gene-editingis carried out after the TILs have been exposed to the OKT-3.
 92. Themethod according to any of claims 65-91, wherein the gene-editing causesexpression of one or more immune checkpoint genes to be silenced orreduced in at least a portion of the therapeutic population of TILs. 93.The method according to claim 92, wherein said one or more immunecheckpoint genes is/are selected from the group comprising PD-1, CTLA-4,LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6,PTPN22, PDCD1, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9,CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD,FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB,HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF,GUCY1A2, GUCY1A3, GUCY1B2, and GUCY1B3.
 94. The method according toclaim 92, wherein said one or more immune checkpoint genes is/areselected from the group comprising PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3),Cish, TGFβ, and PKA.
 95. The method according to any of claims 65-91,wherein the gene-editing causes expression of one or more immunecheckpoint genes to be enhanced in at least a portion of the therapeuticpopulation of TILs, the immune checkpoint gene(s) being selected fromthe group comprising CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL-4,IL-7, IL-10, IL-15, IL-21, the NOTCH 1/2 intracellular domain (ICD),and/or the NOTCH ligand mDLL1.
 96. The method according to any of claims65-95, wherein the gene-editing comprises the use of a programmablenuclease that mediates the generation of a double-strand orsingle-strand break at said one or more immune checkpoint genes.
 97. Themethod according to any of claims 65-95, wherein the gene-editingcomprises one or more methods selected from a CRISPR method, a TALEmethod, a zinc finger method, and a combination thereof.
 98. The methodaccording to any of claims 65-95, wherein the gene-editing comprises aCRISPR method.
 99. The method according to claim 98, wherein the CRISPRmethod is a CRISPR/Cas9 method.
 100. The method according to any ofclaims 65-95, wherein the gene-editing comprises a TALE method.
 101. Themethod according to any of claims 65-95, wherein the gene-editingcomprises a zinc finger method.
 102. A method for expanding tumorinfiltrating lymphocytes (TILs) into a therapeutic population of TILscomprising: (a) adding processed tumor fragments from a tumor resectedfrom a patient into a closed system to obtain a first population ofTILs; (b) performing a first expansion by culturing the first populationof TILs in a cell culture medium comprising IL-2, and optionally OKT-3,to produce a second population of TILs, wherein the first expansion isperformed in a closed container providing a first gas-permeable surfacearea, wherein the first expansion is performed for about 3-14 days toobtain the second population of TILs, wherein the second population ofTILs is at least 50-fold greater in number than the first population ofTILs, and wherein the transition from step (a) to step (b) occurswithout opening the system; (c) performing a second expansion bysupplementing the cell culture medium of the second population of TILswith additional IL-2, optionally OKT-3, and antigen presenting cells(APCs), to produce a third population of TILs, wherein the secondexpansion is performed for about 7-14 days to obtain the thirdpopulation of TILs, wherein the third population of TILs is atherapeutic population of TILs, wherein the second expansion isperformed in a closed container providing a second gas-permeable surfacearea, and wherein the transition from step (b) to step (c) occurswithout opening the system; (d) harvesting the therapeutic population ofTILs obtained from step (c), wherein the transition from step (c) tostep (d) occurs without opening the system; (e) transferring theharvested TIL population from step (d) to an infusion bag, wherein thetransfer from step (d) to (e) occurs without opening the system; and (f)at any time during the method, gene-editing at least a portion of theTILs.
 103. The method according to claim 102, wherein the therapeuticpopulation of TILs harvested in step (d) comprises sufficient TILs for atherapeutically effective dosage of the TILs.
 104. The method accordingto claim 103, where the number of TILs sufficient for a therapeuticallyeffective dosage is from about 2.3×10¹⁰ to about 13.7×10¹⁰.
 105. Themethod according to claim 104, further comprising the step ofcryopreserving the infusion bag comprising the harvested TIL populationusing a cryopreservation process.
 106. The method according to claim105, wherein the cryopreservation process is performed using a 1:1 ratioof harvested TIL population to cryopreservation media.
 107. The methodaccording to claim 102, wherein the antigen-presenting cells areperipheral blood mononuclear cells (PBMCs).
 108. The method according toclaim 107, wherein the PBMCs are irradiated and allogeneic.
 109. Themethod according to claim 108, wherein the PBMCs are added to the cellculture on any of days 9 through 14 in step (c).
 110. The methodaccording to claim 102, wherein the antigen-presenting cells areartificial antigen-presenting cells.
 111. The method according to claim102, wherein the harvesting in step (d) is performed using a LOVO cellprocessing system.
 112. The method according to claim 62, wherein themultiple fragments comprise about 4 to about 50 fragments, wherein eachfragment has a volume of about 27 mm³.
 113. The method according toclaim 102, wherein the multiple fragments comprise about 30 to about 60fragments with a total volume of about 1300 mm³ to about 1500 mm³. 114.The method according to claim 103, wherein the multiple fragmentscomprise about 50 fragments with a total volume of about 1350 mm³. 115.The method according to claim 102, wherein the multiple fragmentscomprise about 50 fragments with a total mass of about 1 gram to about1.5 grams.
 116. The method according to claim 102, wherein the multiplefragments comprise about 4 fragments.
 117. The method according to claim102, wherein the second cell culture medium is provided in a containerselected from the group consisting of a G-container and a Xuri cellbag.118. The method according to claim 102, wherein the infusion bag in step(e) is a HypoThermosol-containing infusion bag.
 119. The methodaccording to claim 102, wherein the first period in step (b) and thesecond period in step (c) are each individually performed within aperiod of 10 days, 11 days, or 12 days.
 120. The method according toclaim 102, wherein the first period in step (b) and the second period instep (c) are each individually performed within a period of 11 days.121. The method according to claim 102, wherein steps (a) through (e)are performed within a period of about 10 days to about 22 days. 122.The method according to claim 102, wherein steps (a) through (e) areperformed within a period of about 10 days to about 20 days.
 123. Themethod according to claim 102, wherein steps (a) through (e) areperformed within a period of about 10 days to about 15 days.
 124. Themethod according to claim 102, wherein steps (a) through (e) areperformed in 22 days or less.
 125. The method according to claim 105,wherein steps (a) through (e) and cryopreservation are performed in 22days or less.
 126. The method according to any one of claims 102 to 125,wherein steps (b) through (e) are performed in a single container,wherein performing steps (b) through (e) in a single container resultsin an increase in TIL yield per resected tumor as compared to performingsteps (b) through (e) in more than one container.
 127. The methodaccording to any one of claims 102 to 126, wherein theantigen-presenting cells are added to the TILs during the second periodin step (c) without opening the system.
 128. The method according to anyone of claims 102 to 127, wherein the third population of TILs in step(d) is a therapeutic population of TILs which comprises an increasedsubpopulation of effector T cells and/or central memory T cells relativeto the second population of TILs, wherein the effector T cells and/orcentral memory T cells obtained in the therapeutic population of TILsexhibit one or more characteristics selected from the group consistingof expressing CD27+, expressing CD28+, longer telomeres, increased CD57expression, and decreased CD56 expression relative to effector T cells,and/or central memory T cells obtained from the second population ofcells.
 129. The method according to any one of claims 102 to 128,wherein the effector T cells and/or central memory T cells obtained inthe therapeutic population of TILs exhibit increased CD57 expression anddecreased CD56 expression relative to effector T cells, and/or centralmemory T cells obtained from the second population of cells.
 130. Themethod according to any one of claims 102 to 129, wherein the risk ofmicrobial contamination is reduced as compared to an open system. 131.The method according to any one of claims 102 to 130, wherein the TILsfrom step (e) are infused into a patient.
 132. The method according toany of claims 102 to 131 wherein the closed container comprises a singlebioreactor.
 133. The method according to claim 132, wherein the closedcontainer comprises a G-REX-10.
 134. The method according to claim 132,wherein the closed container comprises a G-REX-100.
 135. The methodaccording to any one of claims 102 to 134, wherein at step (d) theantigen presenting cells (APCs) are added to the cell culture of thesecond population of TILs at a APC:TIL ratio of 25:1 to 100:1.
 136. Themethod according to claim 135, wherein the cell culture has a ratio of2.5×10⁹ APCs to 100×10⁶ TILs.
 137. The method according to any one ofclaims 102 to 134, wherein at step (c) the antigen presenting cells(APCs) are added to the cell culture of the second population of TILs ata APC:TIL ratio of 25:1 to 100:1.
 138. The method according to claim137, wherein the cell culture has ratio of 2.5×10⁹ APCs to 100×10⁶ TILs.139. The method according to any one of claims 102-138, wherein the cellculture medium further comprises a 4-1BB agonist and/or an OX40 agonistduring the first expansion, the second expansion, or both.
 140. Themethod according to claim 139, wherein and the gene-editing is carriedout after the 4-1BB agonist and/or the OX40 agonist is introduced intothe cell culture medium.
 141. The method according to claim 139, whereinand the gene-editing is carried out before the 4-1BB agonist and/or theOX40 agonist is introduced into the cell culture medium.
 142. The methodaccording to any of claims 102-141, wherein the gene-editing is carriedout on TILs from one or more of the first population, the secondpopulation, and the third population.
 143. The method according to anyof claims 102-141, wherein the gene-editing is carried out on TILs fromthe first expansion, or TILs from the second expansion, or both. 144.The method according to any of claims 102-141, wherein the gene-editingis carried out after the first expansion and before the secondexpansion.
 145. The method according to any of claims 102-141, whereinthe gene-editing is carried out before step (b), before step (c), orbefore step (d).
 146. The method according to any of claims 102-145,wherein the cell culture medium comprises OKT-3 during the firstexpansion and/or during the second expansion, and the gene-editing iscarried out before the OKT-3 is introduced into the cell culture medium.147. The method according to any of claims 102-145, wherein the cellculture medium comprises OKT-3 during the first expansion and/or duringthe second expansion, and the gene-editing is carried out after theOKT-3 is introduced into the cell culture medium.
 148. The methodaccording to any of claims 102-145, wherein the cell culture mediumcomprises OKT-3 beginning on the start day of the first expansion, andthe gene-editing is carried out after the TILs have been exposed to theOKT-3.
 149. The method according to any of claims 102-148, wherein thegene-editing causes expression of one or more immune checkpoint genes tobe silenced or reduced in at least a portion of the therapeuticpopulation of TILs.
 150. The method according to claim 149, wherein saidone or more immune checkpoint genes is/are selected from the groupcomprising PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B,PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, BTLA, CD160, TIGIT, CD96, CRTAM,LAIR1, SIGLEC7, SIGLEC9, CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10,CASP3, CASP6, CASP7, FADD, FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL,TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1,FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, and GUCY1B3.
 151. Themethod according to claim 149, wherein said one or more immunecheckpoint genes is/are selected from the group comprising PD-1, CTLA-4,LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, and PKA.
 152. The method according toany of claims 102-148, wherein the gene-editing causes expression of oneor more immune checkpoint genes to be enhanced in at least a portion ofthe therapeutic population of TILs, the immune checkpoint gene(s) beingselected from the group comprising CCR2, CCR4, CCR5, CXCR2, CXCR3,CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-21, the NOTCH 1/2intracellular domain (ICD), and/or the NOTCH ligand mDLL1.
 153. Themethod according to any of claims 102-152, wherein the gene-editingcomprises the use of a programmable nuclease that mediates thegeneration of a double-strand or single-strand break at said one or moreimmune checkpoint genes.
 154. The method according to any of claims102-152, wherein the gene-editing comprises one or more methods selectedfrom a CRISPR method, a TALE method, a zinc finger method, and acombination thereof.
 155. The method according to any of claims 102-152,wherein the gene-editing comprises a CRISPR method.
 156. The methodaccording to claim 155, wherein the CRISPR method is a CRISPR/Cas9method.
 157. The method according to any of claims 102-152, wherein thegene-editing comprises a TALE method.
 158. The method according to anyof claims 102-152, wherein the gene-editing comprises a zinc fingermethod.
 159. A population of expanded TILs for use in the treatment of asubject with cancer, wherein the population of expanded TILs is a thirdpopulation of TILs obtainable by a method comprising: (a) obtaining afirst population of TILs from a tumor resected from a subject byprocessing a tumor sample obtained from the patient into multiple tumorfragments; (b) adding the tumor fragments into a closed system; (c)performing a first expansion by culturing the first population of TILsin a cell culture medium comprising IL-2, and optionally OKT-3, toproduce a second population of TILs, wherein the first expansion isperformed in a closed container providing a first gas-permeable surfacearea, wherein the first expansion is performed for about 3-14 days toobtain the second population of TILs, wherein the second population ofTILs is at least 50-fold greater in number than the first population ofTILs, and wherein the transition from step (b) to step (c) occurswithout opening the system; (d) performing a second expansion bysupplementing the cell culture medium of the second population of TILswith additional IL-2, optionally OKT-3, and antigen presenting cells(APCs), to produce a third population of TILs, wherein the secondexpansion is performed for about 7-14 days to obtain the thirdpopulation of TILs, wherein the third population of TILs is atherapeutic population of TILs, wherein the second expansion isperformed in a closed container providing a second gas-permeable surfacearea, and wherein the transition from step (c) to step (d) occurswithout opening the system; (e) harvesting the therapeutic population ofTILs obtained from step (d), wherein the transition from step (d) tostep (e) occurs without opening the system; (f) transferring theharvested TIL population from step (e) to an infusion bag, wherein thetransfer from step (e) to (f) occurs without opening the system; (g)optionally cryopreserving the infusion bag comprising the harvested TILpopulation from step (f) using a cryopreservation process; and (h) atany time during the method, gene-editing at least a portion of the TILs.160. The population of TILs for use to treat a subject with canceraccording to claim 159, wherein the method further comprises one or moreof the features recited in any of claims 1 to
 158. 161. The populationof TILs for use to treat a subject with cancer according to claim 159,wherein the cell culture medium further comprises a 4-1BB agonist and/oran OX40 agonist during the first expansion, the second expansion, orboth.
 162. The population of TILs for use to treat a subject with canceraccording to claim 159, wherein the gene-editing is carried out on TILsfrom one or more of the first population, the second population, and thethird population.
 163. The population of TILs for use to treat a subjectwith cancer according to claim 159, wherein the gene-editing is carriedout on TILs from the first expansion, or TILs from the second expansion,or both.
 164. The population of TILs for use to treat a subject withcancer according to claim 159, wherein the gene-editing is carried outafter the first expansion and before the second expansion.
 165. Thepopulation of TILs for use to treat a subject with cancer according toclaim 159, wherein the gene-editing is carried out before step (c),before step (d), or before step (e)
 166. The population of TILs for useto treat a subject with cancer according to claim 159, wherein thegene-editing causes expression of one or more immune checkpoint genes tobe silenced or reduced in at least a portion of the population ofexpanded TILs.
 167. The population of TILs for use to treat a subjectwith cancer according to claim 166, wherein said one or more immunecheckpoint genes is/are selected from the group comprising PD-1, CTLA-4,LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, PKA, CBL-B, PPP2CA, PPP2CB, PTPN6,PTPN22, PDCD1, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9,CD244, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD,FAS, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB,HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF,GUCY1A2, GUCY1A3, GUCY1B2, and GUCY1B3.
 168. The population of TILs foruse to treat a subject with cancer according to claim 166, wherein saidone or more immune checkpoint genes is/are selected from the groupcomprising PD-1, CTLA-4, LAG-3, HAVCR2 (TIM-3), Cish, TGFβ, and PKA.169. The population of TILs for use to treat a subject with canceraccording to claim 166, wherein the gene-editing causes expression ofone or more immune checkpoint genes to be enhanced in at least a portionof the therapeutic population of TILs, the immune checkpoint gene(s)being selected from the group comprising CCR2, CCR4, CCR5, CXCR2, CXCR3,CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-21, the NOTCH 1/2intracellular domain (ICD), and/or the NOTCH ligand mDLL1.
 170. Thepopulation of TILs for use to treat a subject with cancer according toclaim 159, wherein the gene-editing comprises the use of a programmablenuclease that mediates the generation of a double-strand orsingle-strand break at said one or more immune checkpoint genes. 171.The population of TILs for use to treat a subject with cancer accordingto claim 159, wherein the gene-editing comprises one or more methodsselected from a CRISPR method, a TALE method, a zinc finger method, anda combination thereof.
 172. A cryopreservation composition comprisingthe population of TILs for use to treat a subject with cancer accordingto claim 159, a cryoprotectant medium comprising DMSO, and anelectrolyte solution.
 173. The cryopreservation composition of claim 172further comprising one or more stabilizers and one or more lymphocytegrowth factors.
 174. The cryopreservation composition of claim 173,wherein the one or more stabilizers comprise Human Serum Albumin (HSA)and the one or more lymphocyte growth factors comprise IL-2.
 175. Thecryopreservation composition of claim 174, wherein the cryoprotectantmedium comprising DMSO and the electrolyte solution are present in aratio of about 1.1:1 to about 1:1.1.
 176. The cryopreservationcomposition of claim 174 comprising the population of TILs in an amountof about 1×10⁶ to about 9×10¹⁴, the cryoprotectant medium comprisingDMSO in an amount of about 30 mL to about 70 mL, the electrolytesolution in an amount of about 30 mL to about 70 mL, HSA in an amount ofabout 0.1 g to about 1.0 g, and IL-2 in an amount of about 0.001 mg toabout 0.005 mg.
 177. A method for expanding tumor infiltratinglymphocytes (TILs) into a therapeutic population of TILs comprising: (a)obtaining a first population of TILs from a tumor resected from apatient by processing a tumor sample obtained from the patient intomultiple tumor fragments; (b) adding the tumor fragments into a closedsystem; (c) performing a first expansion by culturing the firstpopulation of TILs in a cell culture medium comprising IL-2 andoptionally comprising OKT-3 and/or a 4-1BB agonist antibody for about 2to 5 days; (d) optionally adding OKT-3, to produce a second populationof TILs, wherein the first expansion is performed in a closed containerproviding a first gas-permeable surface area, wherein the firstexpansion is performed for about 1 to 3 days to obtain the secondpopulation of TILs, wherein the second population of TILs is at least50-fold greater in number than the first population of TILs, and whereinthe transition from step (c) to step (d) occurs without opening thesystem; (e) performing a sterile electroporation step on the secondpopulation of TILs, wherein the sterile electroporation step mediatesthe transfer of at least one gene editor; (f) resting the secondpopulation of TILs for about 1 day; (g) performing a second expansion bysupplementing the cell culture medium of the second population of TILswith additional IL-2, optionally OKT-3 antibody, optionally an OX40antibody, and antigen presenting cells (APCs), to produce a thirdpopulation of TILs, wherein the second expansion is performed for about7 to 11 days to obtain the third population of TILs, wherein the secondexpansion is performed in a closed container providing a secondgas-permeable surface area, and wherein the transition from step (f) tostep (g) occurs without opening the system; (h) harvesting thetherapeutic population of TILs obtained from step (g) to provide aharvested TIL population, wherein the transition from step (g) to step(h) occurs without opening the system, wherein the harvested populationof TILs is a therapeutic population of TILs; (i) transferring theharvested TIL population to an infusion bag, wherein the transfer fromstep (h) to (i) occurs without opening the system; and (j)cryopreserving the harvested TIL population using adimethylsulfoxide-based cryopreservation medium, wherein theelectroporation step comprises the delivery of a Clustered RegularlyInterspersed Short Palindromic Repeat (CRISPR) system, a TranscriptionActivator-Like Effector (TALE) system, or a zinc finger system forinhibiting the expression of a molecule selected from the groupconsisting of PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGFβR2, PRA,CBLB, BAFF (BR3), and combinations thereof.
 178. The method according toclaim 177 comprising performing the first expansion by culturing thefirst population of TILs in a cell culture medium comprising IL-2, OKT-3and a 4-1BB agonist antibody, wherein the OKT-3 and the 4-1BB agonistantibody are optionally present in the cell culture medium beginning onDay 0 or Day
 1. 179. The method according to claim 177 or claim 178,wherein the electroporation step comprises the delivery of a ClusteredRegularly Interspersed Short Palindromic Repeat (CRISPR) system, aTranscription Activator-Like Effector (TALE) system, or a zinc fingersystem for inhibiting the expression of a molecule selected from thegroup consisting of PD-1, LAG-3, TIM-3, CISH, and CBLB, and combinationsthereof.
 180. The method according to claim 177 or claim 178, whereinthe electroporation step comprises the delivery of a Clustered RegularlyInterspersed Short Palindromic Repeat (CRISPR) system, a TranscriptionActivator-Like Effector (TALE) system, or a zinc finger system forinhibiting the expression of PD-1.
 181. The method according to claim177 or claim 178, wherein the electroporation step comprises thedelivery of a Clustered Regularly Interspersed Short Palindromic Repeat(CRISPR) system, a Transcription Activator-Like Effector (TALE) system,or a zinc finger system for inhibiting the expression of LAG-3.
 182. Themethod according to claim 177 or claim 178, wherein the electroporationstep comprises the delivery of a Clustered Regularly Interspersed ShortPalindromic Repeat (CRISPR) system, a Transcription Activator-LikeEffector (TALE) system, or a zinc finger system for inhibiting theexpression of TIM-3.
 183. The method according to claim 177 or claim178, wherein the electroporation step comprises the delivery of aClustered Regularly Interspersed Short Palindromic Repeat (CRISPR)system, a Transcription Activator-Like Effector (TALE) system, or a zincfinger system for inhibiting the expression of CISH.
 184. The methodaccording to claim 177 or claim 178, wherein the electroporation stepcomprises the delivery of a Clustered Regularly Interspersed ShortPalindromic Repeat (CRISPR) system, a Transcription Activator-LikeEffector (TALE) system, or a zinc finger system for inhibiting theexpression of CBLB.
 185. The method according to any of claims 177 to184, wherein the electroporation step comprises the delivery of aClustered Regularly Interspersed Short Palindromic Repeat (CRISPR)system.
 186. The method according to any of claims 177 to 184, whereinthe electroporation step comprises the delivery of a TranscriptionActivator-Like Effector (TALE) system.
 187. The method according to anyof claims 177 to 184, wherein the electroporation step comprises thedelivery of a zinc finger system.
 188. The method according to any ofclaims 177 to 187, wherein the dimethylsulfoxide-based cryopreservationmedium comprises DMSO, an electrolyte solution, optionally HSA, andoptionally IL-2.
 189. A method for treating a subject with cancer, themethod comprising administering expanded tumor infiltrating lymphocytes(TILs) comprising: (a) obtaining a first population of TILs from a tumorresected from a patient by processing a tumor sample obtained from thepatient into multiple tumor fragments; (b) adding the tumor fragmentsinto a closed system; (c) performing a first expansion by culturing thefirst population of TILs in a cell culture medium comprising IL-2 andoptionally comprising OKT-3 and/or a 4-1BB agonist antibody for about 2to 5 days; (d) optionally adding OKT-3, to produce a second populationof TILs, wherein the first expansion is performed in a closed containerproviding a first gas-permeable surface area, wherein the firstexpansion is performed for about 1 to 3 days to obtain the secondpopulation of TILs, wherein the second population of TILs is at least50-fold greater in number than the first population of TILs, and whereinthe transition from step (c) to step (d) occurs without opening thesystem; (e) performing a sterile electroporation step on the secondpopulation of TILs, wherein the sterile electroporation step mediatesthe transfer of at least one gene editor; (f) resting the secondpopulation of TILs for about 1 day; (g) performing a second expansion bysupplementing the cell culture medium of the second population of TILswith additional IL-2, optionally OKT-3 antibody, optionally an OX40antibody, and antigen presenting cells (APCs), to produce a thirdpopulation of TILs, wherein the second expansion is performed for about7 to 11 days to obtain the third population of TILs, wherein the secondexpansion is performed in a closed container providing a secondgas-permeable surface area, and wherein the transition from step (f) tostep (g) occurs without opening the system; (h) harvesting thetherapeutic population of TILs obtained from step (g) to provide aharvested TIL population, wherein the transition from step (g) to step(h) occurs without opening the system, wherein the harvested populationof TILs is a therapeutic population of TILs; (i) transferring theharvested TIL population to an infusion bag, wherein the transfer fromstep (h) to (i) occurs without opening the system; (j) cryopreservingthe harvested TIL population using a dimethylsulfoxide-basedcryopreservation medium; and (k) administering a therapeuticallyeffective dosage of the harvested TIL population from the infusion bagto the patient; wherein the electroporation step comprises the deliveryof a Clustered Regularly Interspersed Short Palindromic Repeat (CRISPR)system, a Transcription Activator-Like Effector (TALE) system, or a zincfinger system for inhibiting the expression of a molecule selected fromthe group consisting of PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGFβR2,PRA, CBLB, BAFF (BR3), and combinations thereof.
 190. The methodaccording to claim 189 comprising performing the first expansion byculturing the first population of TILs in a cell culture mediumcomprising IL-2, OKT-3 and a 4-1BB agonist antibody, wherein the OKT-3and the 4-1BB agonist antibody are optionally present in the cellculture medium beginning on Day 0 or Day
 1. 191. The method according toclaim 189 or claim 190, wherein the electroporation step comprises thedelivery of a Clustered Regularly Interspersed Short Palindromic Repeat(CRISPR) system, a Transcription Activator-Like Effector (TALE) system,or a zinc finger system for inhibiting the expression of a moleculeselected from the group consisting of PD-1, LAG-3, TIM-3, CISH, andCBLB, and combinations thereof.
 192. The method according to claim 189or claim 190, wherein the electroporation step comprises the delivery ofa Clustered Regularly Interspersed Short Palindromic Repeat (CRISPR)system, a Transcription Activator-Like Effector (TALE) system, or a zincfinger system for inhibiting the expression of PD-1.
 193. The methodaccording to claim 189 or claim 190, wherein the electroporation stepcomprises the delivery of a Clustered Regularly Interspersed ShortPalindromic Repeat (CRISPR) system, a Transcription Activator-LikeEffector (TALE) system, or a zinc finger system for inhibiting theexpression of LAG-3.
 194. The method according to claim 189 or claim190, wherein the electroporation step comprises the delivery of aClustered Regularly Interspersed Short Palindromic Repeat (CRISPR)system, a Transcription Activator-Like Effector (TALE) system, or a zincfinger system for inhibiting the expression of TIM-3.
 195. The methodaccording to claim 189 or claim 190, wherein the electroporation stepcomprises the delivery of a Clustered Regularly Interspersed ShortPalindromic Repeat (CRISPR) system, a Transcription Activator-LikeEffector (TALE) system, or a zinc finger system for inhibiting theexpression of CISH.
 196. The method according to claim 189 or claim 190,wherein the electroporation step comprises the delivery of a ClusteredRegularly Interspersed Short Palindromic Repeat (CRISPR) system, aTranscription Activator-Like Effector (TALE) system, or a zinc fingersystem for inhibiting the expression of CBLB.
 197. The method accordingto any of claims 189 to 196, wherein the electroporation step comprisesthe delivery of a Clustered Regularly Interspersed Short PalindromicRepeat (CRISPR) system.
 198. The method according to any of claims 189to 196, wherein the electroporation step comprises the delivery of aTranscription Activator-Like Effector (TALE) system.
 199. The methodaccording to any of claims 189 to 196, wherein the electroporation stepcomprises the delivery of a zinc finger system.
 200. The methodaccording to any of claims 189 to 196, wherein thedimethylsulfoxide-based cryopreservation medium comprises DMSO, anelectrolyte solution, optionally HSA, and optionally IL-2.
 201. Apopulation of expanded TILs for use in the treatment of a subject withcancer, wherein the population of expanded TILs is a harvestedpopulation of TILs obtainable by a method comprising: (a) obtaining afirst population of TILs from a tumor resected from a patient byprocessing a tumor sample obtained from the patient into multiple tumorfragments; (b) adding the tumor fragments into a closed system; (c)performing a first expansion by culturing the first population of TILsin a cell culture medium comprising IL-2 and optionally comprising OKT-3and/or a 4-1BB agonist antibody for about 2 to 5 days; (d) optionallyadding OKT-3, to produce a second population of TILs, wherein the firstexpansion is performed in a closed container providing a firstgas-permeable surface area, wherein the first expansion is performed forabout 1 to 3 days to obtain the second population of TILs, wherein thesecond population of TILs is at least 50-fold greater in number than thefirst population of TILs, and wherein the transition from step (c) tostep (d) occurs without opening the system; (e) performing a sterileelectroporation step on the second population of TILs, wherein thesterile electroporation step mediates the transfer of at least one geneeditor; (f) resting the second population of TILs for about 1 day; (g)performing a second expansion by supplementing the cell culture mediumof the second population of TILs with additional IL-2, optionally OKT-3antibody, optionally an OX40 antibody, and antigen presenting cells(APCs), to produce a third population of TILs, wherein the secondexpansion is performed for about 7 to 11 days to obtain the thirdpopulation of TILs, wherein the second expansion is performed in aclosed container providing a second gas-permeable surface area, andwherein the transition from step (f) to step (g) occurs without openingthe system; (h) harvesting the therapeutic population of TILs obtainedfrom step (g) to provide a harvested TIL population, wherein thetransition from step (g) to step (h) occurs without opening the system,wherein the harvested population of TILs is a therapeutic population ofTILs; (i) transferring the harvested TIL population to an infusion bag,wherein the transfer from step (h) to (i) occurs without opening thesystem; and (j) cryopreserving the harvested TIL population using adimethylsulfoxide-based cryopreservation medium, wherein theelectroporation step comprises the delivery of a Clustered RegularlyInterspersed Short Palindromic Repeat (CRISPR) system, a TranscriptionActivator-Like Effector (TALE) system, or a zinc finger system forinhibiting the expression of a molecule selected from the groupconsisting of PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGFβR2, PRA,CBLB, BAFF (BR3), and combinations thereof.
 202. The population ofexpanded TILs according to claim 201 comprising performing the firstexpansion by culturing the first population of TILs in a cell culturemedium comprising IL-2, OKT-3 and a 4-1BB agonist antibody, wherein theOKT-3 and the 4-1BB agonist antibody are optionally present in the cellculture medium beginning on Day 0 or Day
 1. 203. The population ofexpanded TILs according to claim 201 or claim 202, wherein theelectroporation step comprises the delivery of a Clustered RegularlyInterspersed Short Palindromic Repeat (CRISPR) system, a TranscriptionActivator-Like Effector (TALE) system, or a zinc finger system forinhibiting the expression of a molecule selected from the groupconsisting of PD-1, LAG-3, TIM-3, CISH, and CBLB, and combinationsthereof.
 204. The population of expanded TILs according to claim 201 orclaim 202, wherein the electroporation step comprises the delivery of aClustered Regularly Interspersed Short Palindromic Repeat (CRISPR)system, a Transcription Activator-Like Effector (TALE) system, or a zincfinger system for inhibiting the expression of PD-1.
 205. The populationof expanded TILs according to claim 201 or claim 202, wherein theelectroporation step comprises the delivery of a Clustered RegularlyInterspersed Short Palindromic Repeat (CRISPR) system, a TranscriptionActivator-Like Effector (TALE) system, or a zinc finger system forinhibiting the expression of LAG-3.
 206. The population of expanded TILsaccording to claim 201 or claim 202, wherein the electroporation stepcomprises the delivery of a Clustered Regularly Interspersed ShortPalindromic Repeat (CRISPR) system, a Transcription Activator-LikeEffector (TALE) system, or a zinc finger system for inhibiting theexpression of TIM-3.
 207. The population of expanded TILs according toclaim 201 or claim 202, wherein the electroporation step comprises thedelivery of a Clustered Regularly Interspersed Short Palindromic Repeat(CRISPR) system, a Transcription Activator-Like Effector (TALE) system,or a zinc finger system for inhibiting the expression of CISH.
 208. Thepopulation of expanded TILs according to claim 201 or claim 202, whereinthe electroporation step comprises the delivery of a Clustered RegularlyInterspersed Short Palindromic Repeat (CRISPR) system, a TranscriptionActivator-Like Effector (TALE) system, or a zinc finger system forinhibiting the expression of CBLB.
 209. The population of expanded TILsaccording to claim 201 to 208, wherein the electroporation stepcomprises the delivery of a Clustered Regularly Interspersed ShortPalindromic Repeat (CRISPR) system.
 210. The population of expanded TILsaccording to claim 201 to 208, wherein the electroporation stepcomprises the delivery of a Transcription Activator-Like Effector (TALE)system.
 211. The population of expanded TILs according to claim 201 to208, wherein the electroporation step comprises the delivery of a zincfinger system.
 212. The population of expanded TILs according to claim201 to 211, wherein the dimethylsulfoxide-based cryopreservation mediumcomprises DMSO, an electrolyte solution, optionally HSA, and optionallyIL-2.
 213. The population of expanded TILs according to claim 201 to211, wherein the electroporation step comprises a pulsed electroporationstep.
 214. The method according to any of claims 1-58, wherein thegene-editing comprises a CRISPR method, and the CRISPR method includesuse of a high-fidelity Cas9.